Salt formulations for the fermentation of marine microorganisms

ABSTRACT

Growth medium are disclosed for use in fermenting a marine microorganism. The medium comprise Potassium, Calcium, Strontium, Borate and Fluoride at specific concentrations. Alternatively, the growth medium comprises cobalt at specified concentrations or comprises vitamin B 12  at specified concentrations. Methods of producing certain desired compound by fermentation of a marine microorganism are also disclosed.

RELATED APPLICATIONS

The application claims priority to U.S. Provisional Application No.60/846,774, filed Sep. 22, 2006, and U.S. Provisional Application No.60/949,147, filed Jul. 11, 2007, and U.S. Provisional Application Nos.60/952,349 and 60/952,368, both filed Jul. 27, 2007. Each of theseapplications is incorporated herein, in its entirety, by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of culturing microorganisms, inparticular to systems, formulations and methods for the production ofmarine-derived microorganisms and compounds that they produce.

2. Description of the Related Art

Cancer is a leading cause of death in the United States. Despitesignificant efforts to find new approaches for treating cancer, theprimary treatment options remain surgery, chemotherapy and radiationtherapy, either alone or in combination. Surgery and radiation therapy,however, are generally useful only for fairly defined types of cancer,and are of limited use for treating patients with disseminated disease.Chemotherapy is the method that is generally useful in treating patientswith metastatic cancer or diffuse cancers such as leukemias. Althoughchemotherapy can provide a therapeutic benefit, it often fails to resultin cure of the disease due to the patient's cancer cells becomingresistant to the chemotherapeutic agent. Due, in part, to the likelihoodof cancer cells becoming resistant to a chemotherapeutic agent, suchagents are commonly used in combination to treat patients.

Similarly, infectious diseases caused, for example, by bacteria, fungiand protozoa are becoming increasingly difficult to treat and cure. Forexample, more and more bacteria, fungi and protozoa are developingresistance to current antibiotics and chemotherapeutic agents. Examplesof such microbes include Bacillus, Leishmania, Plasmodium andTrypanosoma.

Furthermore, a growing number of diseases and medical conditions areclassified as inflammatory diseases. Such diseases include conditionssuch as asthma to cardiovascular diseases. These diseases continue toaffect larger and larger numbers of people worldwide despite newtherapies and medical advances.

Therefore, a need exists for additional chemotherapeutics,anti-microbial agents, and anti-inflammatory agents to treat cancer,inflammatory diseases and infectious disease. A continuing effort isbeing made by individual investigators, academia and companies toidentify new, potentially useful chemotherapeutic and anti-microbialagents.

Marine-derived natural products are a rich source of potential newanti-cancer agents and anti-microbial agents. The oceans are massivelycomplex and house a diverse assemblage of microbes that occur inenvironments of extreme variations in pressure, salinity, andtemperature. Marine microorganisms have therefore developed uniquemetabolic and physiological capabilities that not only ensure survivalin extreme and varied habitats, but also offer the potential to producemetabolites that would not be observed from terrestrial microorganisms(Okami, Y. 1993 J Mar Biotechnol 1:59). Representative structuralclasses of such metabolites include terpenes, peptides, polyketides, andcompounds with mixed biosynthetic origins. Many of these molecules havedemonstrable anti-tumor, anti-bacterial, anti-fungal, anti-inflammatoryor immunosuppressive activities (Bull, A. T. et al. 2000 Microbiol MolBiol Rev 64:573; Cragg, G. M. & D. J. Newman 2002 Trends Pharmacol Sci23:404; Kerr, R. G. & S. S. Kerr 1999 Exp Opin Ther Patents 9:1207;Moore, B. S 1999 Nat Prod Rep 16:653; Faulkner, D. J. 2001 Nat Prod Rep18:1; Mayer, A. M. & V. K. Lehmann 2001 Anticancer Res 21:2489),validating the utility of this source for isolating invaluabletherapeutic agents. Further, the isolation of novel anti-cancer andanti-microbial agents that represent alternative mechanistic classes tothose currently on the market will help to address resistance concerns,including any mechanism-based resistance that may have been engineeredinto pathogens for bioterrorism purposes.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a growth medium for use infermenting a marine microorganism, the medium comprising (a) Potassiumion at a concentration between 5 mMole/L and 13 mMole/L; (b) Calciumspecies at a concentration between 1.8 mMole/L and 6.3 mMole/L; (c)Strontium ion at a concentration between 0.03 mMole/L and 0.32 mMole/L;(d) Borate species at a concentration between 0.16 mMole/L and 0.81mMole/L; and (e) Fluoride ion at a concentration between 0.02 mMole/Land 0.11 mMole/L.

In another embodiment, the invention relates to a growth medium for usein fermenting a marine microorganism, the medium comprising cobalt ionat a concentration between 0.2 μMole/L and 1.9 μMole/L. In anotherembodiment, this medium comprises vitamin B₁₂ at a concentration between0.05 mg/L and 5 mg/L.

In another embodiment, the invention relates to a salt formulation foruse in fermenting a marine microorganism, the formulation comprising:NaCl in the salt formulation at a mass percent between 96% and 80%; KClin the salt formulation at a mass percent between 5% and 1.50%; CaCl₂ inthe salt formulation at a mass percent between 3% and 1%; SrCl₂ in thesalt formulation at a mass percent between 0.10% and 0.025%; H₃BO₃ inthe salt formulation at a mass percent between 0.20% and 0.04%; and NaFin the salt formulation at a mass percent between 0.05% and 0.005%.

In another embodiment, the invention relates to a salt formulation foruse in fermenting a marine microorganism, the formulation comprising:Na₂SO₄ at a mass percent between 95% and 90%; KCl at a mass percentbetween 8% and 6%; CaCl₂ at a mass percent between 5% and 3%; SrCl₂ at amass percent between 0.15% and 0.05%; H₃BO₃ at a mass percent between0.30% and 0.10%; and NaF at a mass percent between 0.03% and 0.01%.

In another embodiment, the invention relates to a salt formulation foruse in fermenting a marine microorganism, the formulation comprising:KCl at a mass percent between 70% and 50%; CaCl₂ at a mass percentbetween 45% and 30%; SrCl₂ at a mass percent between 3% and 1%; H₃BO₃ ata mass percent between 3% and 1%; and NaF at a mass percent between 0.3%and 0.1%.

In another embodiment, the invention relates to a method of fermenting amarine microorganism, the method comprising:

-   -   (a) preparing a salt solution comprising:        -   (i) KCl concentration between 400 mg/L and 1 g/L;        -   (ii) CaCl₂ concentration between 200 mg/L and 700 mg/L;        -   (iii) SrCl₂ concentration between 5 mg/L and 50 mg/L;        -   (iv) H₃BO₃ concentration between 10 mg/L and 50 mg/L; and        -   (v) NaF concentration between 1 mg/L and 5 mg/L.    -   (b) adding a carbon source;    -   (c) adding a nitrogen source;    -   (d) adding a member of the group consisting of NaCl at a        concentration between 5 g/L and 30 g/L, Na₂SO₄ at a        concentration between 10 g/L and 40 g/L, and KCl at a        concentration between 25 g/L and 35 g/L; and    -   (e) adding the microorganism.

In another embodiment, the invention relates to a method of producing adesired compound by fermentation of a marine microorganism, the methodcomprising adding CoCl₂ to a medium for growing the microorganism,wherein the amount of CoCl₂ added is sufficient to increase the amountof the desired compound produced and reduce the amount of an undesiredcompound produced relative to the amounts produced in the absence ofCoCl₂.

In another embodiment, the invention relates to a method of producingsalinosporamide A, comprising the steps of preparing a microbial growthmedia; inoculating said media with a strain of microorganism capable ofproducing salinosporamide A; adding a resin to the media before 96 hoursafter inoculation of the medium. In another embodiment of the resin isadded before 24 hours after inoculation of the medium.

In another embodiment, the invention relates to a method of producing aproteasome inhibitor comprising culturing Salinospora tropica in thepresence of Amberlite XAD-7 resin.

In another embodiment, the invention relates to a method of producing acompound of Formula A, wherein R1 is selected from the group consistingof hydrogen and hydroxide, R2 is selected from the group consisting of2-chloroethyl, 2-bromoethyl, ethyl, methyl, and propyl, and R3 isselected from the group consisting of methyl and ethyl, comprising thesteps of growing a culture of S. tropica in the presence of XAD-7 resin.

In another embodiment, the invention relates to a method of producing acompound of Formula B, wherein R1 is selected from the group consistingof hydrogen and hydroxide, R2 is selected from the group consisting of2-chloroethyl, 2-bromoethyl, ethyl, methyl, and propyl, and R3 isselected from the group consisting of methyl and ethyl, comprising thesteps of growing a culture of S. tropica in the presence of XAD-7 resin.

In another embodiment, the invention relates to a method of producing acompound of Formula C, wherein R2 is selected from the group consistingof 2-chloroethyl and methyl, comprising the steps of growing a cultureof S. tropica in the presence of XAD-7 resin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The production of marine derived natural products can be carried out bycultivating the microorganism that produces the compound in a suitablenutrient medium, in either aerobic or anaerobic conditions, until asubstantial amount of desired compound is detected in the media. Thecompound can then be harvested from the media with a suitable solvent,concentrating the desired compound.

Various separation techniques, well known in the art, can be used toisolate the desired compound, separating it from other metabolites alsopresent in the cultivation medium. Separation of the desired compoundfrom other co-harvested metabolites leads to a decrease in the net yieldof the desired compound. The reduction of other metabolites produced bythe microorganism would result in a higher net yield of the desiredcompound due to decreased loss during the separation process. Reductionof other metabolites produced by the microorganism may also result inincreased production of the desired metabolite.

It has been discovered that varying the concentrations of particularsalts in the growth media of microorganisms may reduce the production ofanalog metabolites and increase the production of the desired compound.Accordingly, some embodiments include salt formulations that areoptimized to increase production of a desired compound and decreaseproduction of undesired metabolites. Furthermore, commercially availablesea salt formulations have component variability that is unsuitable formeeting the quality control requirements of pharmaceutical manufacture.Accordingly, some embodiments include salt formulations optimized forthe production pharmaceutical agents from marine fermentation.

Growth of marine-derived microorganisms is achieved by the use ofappropriate medium. Broadly, medium suitable to cultivate themicroorganisms includes a carbon source, a nitrogen source, and nutrientinorganic salts. In some cases, a seed culture is first grown in a seedmedium followed by inoculation into a production medium. Broadly, thesources of carbon in the seed and production mediums may includeglucose, fructose, mannose, maltose, galactose, mannitol, and/orglycerol, other sugars and/or sugar alcohols, starches and/or othercarbohydrates, or carbohydrate derivatives such as dextran, cerelose, aswell as complex nutrients such as oat flour, corn meal, millet, corn,and the like. The exact quantity of the carbon source that is utilizedin the medium will depend in part, upon the other ingredients in themedium, but an amount of carbohydrate between 0.5 to 25 percent byweight of the medium can be satisfactorily used, for example. Thesecarbon sources can be used individually or several such carbon sourcescan be combined in the same medium, for example.

The sources of nitrogen include amino acids such as glycine, arginine,threonine, methionine and the like, ammonium salt, as well as complexsources such as yeast extracts, corn steep liquors, distiller solubles,soybean meal, cottonseed meal, fish meal, peptone, and the like. Thevarious sources of nitrogen can be used alone or in combination inamounts ranging from 0.5 to 25 percent by weight of the medium, forexample.

Among the nutrient inorganic salts, which can be incorporated in theculture media, are the customary salts capable of yielding sodium,potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate,and like ions. Also included may be trace metals such as cobalt,manganese, iron, molybdenum, zinc, cadmium, and the like. Some of thesesalts may be part of a vegetative or fermentation media, for example, acommercially available vegetative or fermentation media. Other salts maybe provided in a prepared salt formulation. Commercially available seasalt formulations may include INSTANT OCEAN®, Aquarium Systems, Inc.,Wicklife Ohio. However, in some embodiments, a salt formulation isprovided that is optimized for the production of a pharmaceutical agent.

In one embodiment, a salt formulation is provided comprising NaCl,MgSO₄, KBr, KCl, CaCO₃, CaCl₂, SrCl₂, H₃BO₃, and NaF salts. In oneembodiment, this salt formulation is optimized for use in a seed medium.In one embodiment, the salt formulation is provided as a mixture ofsolid salts. In one such embodiment the percent mass of NaCl in thesolid salt is between 99% and 1%. In another embodiment the percent massof NaCl in the solid salt is between 95% and 10%. In another embodimentthe percent mass of NaCl in the solid salt is between 90% and 30%. Inanother embodiment the percent mass of NaCl in the solid salt is between88% and 60%. In another embodiment the percent mass of NaCl in the solidsalt is between 85% and 75%. In the preferred embodiment the percentmass of NaCl in the solid salt is about 80%. In one embodiment thepercent mass of MgSO₄.7H₂O in the solid salt is between 90% and 0.1%. Inanother embodiment the percent mass of MgSO₄.7H₂O in the solid salt isbetween 70% and 1%. In another embodiment the percent mass of MgSO₄.7H₂Oin the solid salt is between 40% and 5%. In another embodiment thepercent mass of MgSO₄.7H₂O in the solid salt is between 20% and 10%. Inthe preferred embodiment the percent mass of MgSO₄.7H₂O in the solidsalt is about 14%. In one embodiment the percent mass of KBr in thesolid salt is between 10% and 0.01%. In another embodiment the percentmass of KBr in the solid salt is between 5% and 0.05%. In anotherembodiment the percent mass of KBr in the solid salt is between 3% and0.10%. In another embodiment the percent mass of KBr in the solid saltis between 1% and 0.20%. In the preferred embodiment the percent mass ofKBr in the solid salt is 0.30%. In one embodiment the percent mass ofKCl in the solid salt is between 90% and 0.10%. In another embodimentthe percent mass of KCl in the solid salt is between 40% and 0.50%. Inanother embodiment the percent mass of KCl in the solid salt is between20% and 1%. In another embodiment the percent mass of KCl in the solidsalt is between 5% and 1.50%. In the preferred embodiment the percentmass of KCl in the solid salt is about 2.5%. In one embodiment thepercent mass of CaCO₃ in the solid salt is between 70% and 0.10%. Inanother embodiment the percent mass of CaCO₃ in the solid salt isbetween 40% and 0.25%. In another embodiment the percent mass of CaCO₃in the solid salt is between 20% and 0.50%. In another embodiment thepercent mass of CaCO₃ in the solid salt is between 5% and 1%. In thepreferred embodiment the percent mass of CaCO₃ in the solid salt isabout 1.5%. In one embodiment the percent mass of CaCl₂ in the solidsalt is between 70% and 0.10%. In another embodiment the percent mass ofCaCl₂ in the solid salt is between 40% and 0.25%. In another embodimentthe percent mass of CaCl₂ in the solid salt is between 20% and 0.50%. Inanother embodiment the percent mass of CaCl₂ in the solid salt isbetween 5% and 1%. In the preferred embodiment the percent mass of CaCl₂in the solid salt is about 1.5%. In one embodiment the percent mass ofSrCl₂ in the solid salt is between 20% and 0.001%. In another embodimentthe percent mass of SrCl₂ in the solid salt is between 5% and 0.005%. Inanother embodiment the percent mass of SrCl₂ in the solid salt isbetween 1% and 0.01%. In another embodiment the percent mass of SrCl₂ inthe solid salt is between 0.10% and 0.025%. In the preferred embodimentthe percent mass of SrCl₂ in the solid salt is about 0.05%. In oneembodiment the percent mass of H₃BO₃ in the solid salt is between 20%and 0.001%. In another embodiment the percent mass of H₃BO₃ in the solidsalt is between 5% and 0.005%. In another embodiment the percent mass ofH₃BO₃ in the solid salt is between 1% and 0.01%. In another embodimentthe percent mass of H₃BO₃ in the solid salt is between 0.10% and 0.04%.In the preferred embodiment the percent mass of H₃BO₃ in the solid saltis about 0.07%. In one embodiment the percent mass of NaF in the solidsalt is between 10% and 0.0001%. In another embodiment the percent massof NaF in the solid salt is between 2% and 0.001%. In another embodimentthe percent mass of NaF in the solid salt is between 0.5% and 0.003%. Inanother embodiment the percent mass of NaF in the solid salt is between0.05% and 0.005%. In the preferred embodiment the percent mass of NaF inthe solid salt is about 0.01%. In one embodiment NaF in the solid saltis replaced by KF in the solid salt between 10% and 0.0001%. In anotherembodiment the percent mass of KF in the solid salt is between 2% and0.001%. In another embodiment the percent mass of KF in the solid saltis between 0.5% and 0.003%. In another embodiment the percent mass of KFin the solid salt is between 0.05% and 0.005%. In the preferredembodiment the percent mass of KF in the solid salt is about 0.01%.

In another embodiment, the salt formulation is provided as a solution inwater. In one such embodiment the NaCl concentration is between 100 mg/Land 200 g/L. In another embodiment the NaCl concentration is between 1g/L and 50 g/L. In another embodiment the NaCl concentration is between10 g/L and 40 g/L. In another embodiment the NaCl concentration isbetween 20 g/L and 30 g/L. In the preferred embodiment the NaClconcentration is about 25 g/L. In one embodiment the MgSO₄.7H₂Oconcentration is between 10 mg/L and 100 g/L. In another embodiment theMgSO₄.7H₂O concentration is between 500 mg/L and 20 g/L. In anotherembodiment the MgSO₄.7H₂O concentration is between 1 g/L and 10 g/L. Inanother embodiment the MgSO₄.7H₂O concentration is between 2 g/L and 6g/L. In the preferred embodiment the MgSO₄.7H₂O concentration is about 4g/L. In one embodiment the KBr concentration is between 1 mg/L and 2g/L. In another embodiment the KBr concentration is between 5 mg/L and400 mg/L. In another embodiment the KBr concentration is between 20 mg/Land 200 mg/L. In another embodiment the KBr concentration is between 70mg/L and 100 mg/L. In the preferred embodiment the KBr concentration isabout 85 mg/L. In one embodiment the KCl concentration is between 10mg/L and 100 g/L. In another embodiment the KCl concentration is between50 mg/L and 20 g/L. In another embodiment the KCl concentration isbetween 200 mg/L and 5 g/L. In another embodiment the KCl concentrationis between 400 mg/L and 1 g/L. In another embodiment the KClconcentration is between 650 mg/L and 750 g/L. In the preferredembodiment the KCl concentration is about 700 mg/L. In one embodimentthe CaCO₃ concentration is between 1 mg/L and 10 g/L. In anotherembodiment the CaCO₃ concentration is between 10 mg/L and 5 g/L. Inanother embodiment the CaCO₃ concentration is between 100 mg/L and 1g/L. In another embodiment the CaCO₃ concentration is between 200 mg/Land 700 mg/L. In another embodiment the CaCO₃ concentration is between400 mg/L and 500 mg/L. In the preferred embodiment the CaCO₃concentration is about 450 mg/L. In one embodiment the CaCl₂concentration is between 1 mg/L and 10 g/L. In another embodiment theCaCl₂ concentration is between 10 mg/L and 5 g/L. In another embodimentthe CaCl₂ concentration is between 100 mg/L and 1 g/L. In anotherembodiment the CaCl₂ concentration is between 200 mg/L and 700 mg/L. Inanother embodiment the CaCl₂ concentration is between 400 mg/L and 500mg/L. In the preferred embodiment the CaCl₂ concentration is about 450mg/L. In one embodiment the SrCl₂ concentration is between 0.1 mg/L and10 g/L. In another embodiment the SrCl₂ concentration is between 0.5mg/L and 1 g/L. In another embodiment the SrCl₂ concentration is between1 mg/L and 500 mg/L. In another embodiment the SrCl₂ concentration isbetween 5 mg/L and 50 mg/L. In another embodiment the SrCl₂concentration is between 10 mg/L and 20 mg/L. In the preferredembodiment the SrCl₂ concentration is about 15 mg/L. In one embodimentthe H₃BO₃ concentration is between 0.1 mg/L and 10 g/L. In anotherembodiment the H₃BO₃ concentration is between 0.5 mg/L and 2 g/L. Inanother embodiment the H₃BO₃ concentration is between 2 mg/L and 500mg/L. In another embodiment the H₃BO₃ concentration is between 10 mg/Land 50 mg/L. In another embodiment the H₃BO₃ concentration is between 15mg/L and 25 mg/L. In the preferred embodiment the H₃BO₃ concentration isabout 20 mg/L. In one embodiment the NaF concentration is between 0.05mg/L and 500 mg/L. In another embodiment the NaF concentration isbetween 0.25 mg/L and 100 mg/L. In another embodiment the NaFconcentration is between 0.50 mg/L and 50 mg/L. In another embodimentthe NaF concentration is between 1 mg/L and 5 mg/L. In the preferredembodiment the NaF concentration is about 3 mg/L. In one embodiment theNaF is replaced by KF using concentration between 0.05 mg/L and 500mg/L. In another embodiment the KF concentration is between 0.25 mg/Land 100 mg/L. In another embodiment the KF concentration is between 0.50mg/L and 50 mg/L. In another embodiment the KF concentration is between1 mg/L and 5 mg/L. In the preferred embodiment the KF concentration isabout 3 mg/L.

In another embodiment, a salt formulation is provided comprising NaCl,KCl, CaCl₂, SrCl₂, H₃BO₃, and NaF salts. In one embodiment, this saltformulation is optimized for use in a production medium. In oneembodiment, the salt formulation is provided as a mixture of solidsalts. In one such embodiment the percent mass of NaCl in the solid saltis between 99.9% and 1%. In another embodiment the percent mass of NaClin the solid salt is between 99% and 10%. In another embodiment thepercent mass of NaCl in the solid salt is between 97% and 50%. Inanother embodiment the percent mass of NaCl in the solid salt is between96% and 80%. In another embodiment the percent mass of NaCl in the solidsalt is between 96% and 90%. In the preferred embodiment the percentmass of NaCl in the solid salt is about 95%. In one embodiment thepercent mass of KCl in the solid salt is between 90% and 0.10%. Inanother embodiment the percent mass of KCl in the solid salt is between40% and 0.50%. In another embodiment the percent mass of KCl in thesolid salt is between 20% and 1%. In another embodiment the percent massof KCl in the solid salt is between 5% and 1.50%. In the preferredembodiment the percent mass of KCl in the solid salt is about 2.5%. Inone embodiment the percent mass of CaCl₂ in the solid salt is between70% and 0.10%. In another embodiment the percent mass of CaCl₂ in thesolid salt is between 40% and 0.25%. In another embodiment the percentmass of CaCl₂ in the solid salt is between 10% and 0.50%. In anotherembodiment the percent mass of CaCl₂ in the solid salt is between 3% and1%. In the preferred embodiment the percent mass of CaCl₂ in the solidsalt is 1.7%. In one embodiment the percent mass of SrCl₂ in the solidsalt is between 20% and 0.001%. In another embodiment the percent massof SrCl₂ in the solid salt is between 5% and 0.005%. In anotherembodiment the percent mass of SrCl₂ in the solid salt is between 1% and0.01%. In another embodiment the percent mass of SrCl₂ in the solid saltis between 0.10% and 0.025%. In the preferred embodiment the percentmass of SrCl₂ in the solid salt is about 0.06%. In one embodiment thepercent mass of H₃BO₃ in the solid salt is between 20% and 0.001%. Inanother embodiment the percent mass of H₃BO₃ in the solid salt isbetween 5% and 0.005%. In another embodiment the percent mass of H₃BO₃in the solid salt is between 1% and 0.01%. In another embodiment thepercent mass of H₃BO₃ in the solid salt is between 0.20% and 0.04%. Inthe preferred embodiment the percent mass of H₃BO₃ in the solid salt isabout 0.09%. In one embodiment the percent mass of NaF in the solid saltis between 10% and 0.0001%. In another embodiment the percent mass ofNaF in the solid salt is between 2% and 0.001%. In another embodimentthe percent mass of NaF in the solid salt is between 0.5% and 0.003%. Inanother embodiment the percent mass of NaF in the solid salt is between0.1% and 0.005%. In the preferred embodiment the percent mass of NaF inthe solid salt is about 0.01%. In one embodiment the percent mass of NaFin the solid salt is replaced by KF between 10% and 0.0001%. In anotherembodiment the percent mass of KF in the solid salt is between 2% and0.001%. In another embodiment the percent mass of KF in the solid saltis between 0.5% and 0.003%. In another embodiment the percent mass of KFin the solid salt is between 0.1% and 0.005%. In the preferredembodiment the percent mass of KF in the solid salt is about 0.01%.

In another embodiment, the salt formulation is provided as a solution inwater. In one such embodiment the NaCl concentration is between 100 mg/Land 200 g/L. In another embodiment the NaCl concentration is between 1g/L and 50 g/L. In another embodiment the NaCl concentration is between10 g/L and 40 g/L. In another embodiment the NaCl concentration isbetween 20 g/L and 30 g/L. In the preferred embodiment the NaClconcentration is about 25 g/L. In one embodiment the KCl concentrationis between 10 mg/L and 100 g/L. In another embodiment the KClconcentration is between 50 mg/L and 20 g/L. In another embodiment theKCl concentration is between 200 mg/L and 5 g/L. In another embodimentthe KCl concentration is between 400 mg/L and 1 g/L. In anotherembodiment the KCl concentration is between 600 mg/L and 800 mg/L. Inthe preferred embodiment the KCl concentration is about 700 mg/L. In oneembodiment the CaCl₂ concentration is between 1 mg/L and 10 g/L. Inanother embodiment the CaCl₂ concentration is between 10 mg/L and 5 g/L.In another embodiment the CaCl₂ concentration is between 100 mg/L and 1g/L. In another embodiment the CaCl₂ concentration is between 200 mg/Land 700 mg/L. In another embodiment the CaCl₂ concentration is between400 mg/L and 500 mg/L. In the preferred embodiment the CaCl₂concentration is about 450 mg/L. In one embodiment the SrCl₂concentration is between 0.1 mg/L and 10 g/L. In another embodiment theSrCl₂ concentration is between 0.5 mg/L and 1 g/L. In another embodimentthe SrCl₂ concentration is between 1 mg/L and 500 mg/L. In anotherembodiment the SrCl₂ concentration is between 5 mg/L and 50 mg/L. Inanother embodiment the SrCl₂ concentration is between 10 mg/L and 20mg/L. In the preferred embodiment the SrCl₂ concentration is about 15mg/L. In one embodiment the H₃BO₃ concentration is between 0.1 mg/L and10 g/L. In another embodiment the H₃BO₃ concentration is between 0.5mg/L and 2 g/L. In another embodiment the H₃BO₃ concentration is between2 mg/L and 500 mg/L. In another embodiment the H₃BO₃ concentration isbetween 10 mg/L and 50 mg/L. In another embodiment the H₃BO₃concentration is between 15 mg/L and 25 mg/L. In the preferredembodiment the H₃BO₃ concentration is about 20 mg/L. In one embodimentthe NaF concentration is between 0.05 mg/L and 500 mg/L. In anotherembodiment the NaF concentration is between 0.25 mg/L and 100 mg/L. Inanother embodiment the NaF concentration is between 0.50 mg/L and 50mg/L. In another embodiment the NaF concentration is between 1 mg/L and5 mg/L. In the preferred embodiment the NaF concentration is about 3mg/L. In one embodiment the NaF concentration is replaced by KF and isbetween 0.05 mg/L and 500 mg/L. In another embodiment the KFconcentration is between 0.25 mg/L and 100 mg/L. In another embodimentthe KF concentration is between 0.50 mg/L and 50 mg/L. In anotherembodiment the KF concentration is between 1 mg/L and 5 mg/L. In thepreferred embodiment the KF concentration is about 3 mg/L.

In some alternative embodiments of the above salt formulations, no NaClis added. Excluding NaCl results in a formulation that is less corrosiveto fermentation vessels. In one embodiment Na₂SO₄ is added at aconcentration between 100 mg/L and 200 g/L to replace NaCl. In anotherembodiment Na₂SO₄ is added at a concentration between 1 g/L and 50 g/Lto replace NaCl. In one embodiment Na₂SO₄ is added at a concentrationbetween 5 g/L and 30 g/L to replace NaCl. In one embodiment Na₂SO₄ isadded at a concentration between 10 g/L and 20 g/L to replace NaCl. Inthe preferred embodiment where Na₂SO₄ is added to replace NaCl, theNa₂SO₄ concentration is about 20 g/L.

In another embodiment of the above salt formulations for seed orproduction media, the sodium ion level is reduced. Embodiments may use,for example, potassium salts or potassium salts and sulfate salts alongwith other nutrient and environmental salts. In one embodiment, KCl isbetween 1 mg/L and 100 g/L. In another embodiment, the KCl is between100 mg/L and 60 g/L. In another embodiment, the KCl is between 2 g/L and45 g/L. In another embodiment MgSO₄ is present in the medium. In oneembodiment, the MgSO₄ is supplied at least in part through addition ofMgSO₄.7H₂O. In one embodiment, the MgSO₄.7H₂O is between 1 mg/L and 20g/L. In another embodiment, the MgSO₄.7H₂O is between 500 mg/L and 15g/L. In another embodiment, the MgSO₄.7H₂O is between 2 and 8 g/L. Inother embodiments, potassium ions and sulfate ions can be supplied bycombining any of the above additions.

In another embodiment of the above salt formulations for seed andproduction media, the chloride ion level is reduced. Embodiments mayuse, for example, sulfate salts. The sulfate salts may be in the sodium,potassium, magnesium or other appropriate forms. The salts may or maynot be hydrated. In various embodiments, Na₂SO₄ is between 10 mg/L and60 g/L or between 500 mg/L and 45 g/L or between 3 g/L and 25 g/L. Inother embodiments, K₂SO₄ is between 10 mg/L and 100 g/L or between 1 g/Land 75 g/L or between 10 g/L and 60 g/L. In other embodiments, lowsodium and low chloride ion levels can be achieved by combiningformulations described above. In other embodiments, a low sodium or lowchloride or low sodium and chloride formulation can be used for only theseed stage, only the production stage, less than all of the seed stages,or a combination of these alternatives.

Another embodiment includes the addition of a cobalt salt to a saltformulation such as the formulations described above. In someembodiments, cobalt salt may be added to a medium utilizing acommercially available salt formulation (e.g., INSTANT OCEAN®). In oneembodiment, the cobalt salt is CoCl₂. In one embodiment the CoCl₂concentration is between 1 μg/L and 20 mg/L. In another embodiment theCoCl₂ concentration is between 5 μg/L and 5 mg/L. In another embodimentthe CoCl₂ concentration is between 10 μg/L and 1 mg/L. In anotherembodiment the CoCl₂ concentration is between 20 μg/L and 250 μg/L. Inthe preferred embodiment the CoCl₂ concentration is between 50 μg/L and210 μg/L.

In one embodiment, the invention relates to a method of producing amarine microorganism, comprising the steps of:

preparing a microbial growth medium;

inoculating said medium with a strain of the microorganism;

adding a resin to the medium within 96 hours after inoculation of themedium.

The microorganism is preferably capable of producing Salinosporamide A.

In certain embodiments, the resin is added within 72 hours afterinoculation of the medium.

In other embodiments, the resin is added within 48 hours afterinoculation of the medium.

In other embodiments, the resin is added within 24 hours of inoculationof the medium.

In other embodiments, the resin is added prior to inoculation of themedium.

In other embodiment, the microorganism is of the genus Salinispora.

In certain embodiments, the microorganism is Salinispora tropica.

In other embodiments, the microorganism is Salinispora tropicaNPS021184.

In another embodiment, the resin is an adsorption resin.

In another embodiment, the resin is an uncharged adsorption resin.

In another embodiment, the resin is a hydrophobic adsorption resin.

In another embodiment, the resin is at least partially composed of amaterial selected from the group consisting ofpolystyrenedivinylbenzene, aliphatic esters, a formophenolic matrix, across-linked styrenic matrix, a brominated cross-linked styrenic matrix,and a methacrylic ester copolymer.

In another embodiment, the resin includes at least one resin selectedfrom the group consisting of Amberlite XAD2, Amberlite XAD4, AmberliteXAD7, Amberlite XAD7HP, Amberlite XAD16, Amberlite XAD761, Amberlite XAD761, Amberlite XAD1180, Diaion HP2MG, Diaion HP20, Diaion HP21,Sepabeads SP825, Sepabeads SP850, Sepabeads SP70, Sepabeads SP700, andSepabeads SP207.

In another embodiment, the resin includes at least one resin selectedfrom the group consisting of Amberlite XAD2, Amberlite XAD4, AmberliteXAD7, Amberlite XAD7HP and Amberlite XAD16.

In another embodiment, the ratio of resin to total nitrogen added to thefermenter is 5:1 to 100:1.

In another embodiment, the ratio of resin to total nitrogen added to thefermenter is 10:1 to 60:1.

In another embodiment, the ratio of resin to total nitrogen added to thefermenter is 20:1 to 40:1.

In another embodiment, the ratio of resin to dry cell weight is 15:1 to120:1.

In another embodiment, the ratio of resin to dry cell weight is 30:1 to45:1.

Another aspect of the invention relates to a method of producingsalinosporamide A, comprising culturing a salinosporamide A producingmicroorganism in the presence of Amberlite XAD-7 resin.

In one embodiment, the ratio of resin to total nitrogen added to thefermenter is 5:1 to 100:1.

In another embodiment, the ratio of resin to total nitrogen added to thefermenter is 10:1 to 60:1.

In another embodiment, the ratio of resin to total nitrogen added to thefermenter is 20:1 to 40:1.

In another embodiment, the ratio of resin to dry cell weight is 15:1 to120:1.

In another embodiment, the ratio of resin to dry cell weight is 30:1 to45:1.

Another aspect of the invention relates to a method of producing aproteasome inhibitor comprising culturing Salinisporamide tropica in thepresence of Amberlite XAD-7 resin.

In one embodiment, XAD-7 resin is added to the fermentation vessel priorto inoculation of the media.

In another embodiment, XAD-7 resin is added after at least a portion ofinoculum is added.

In another embodiment, at least a portion of the XAD-7 resin is addedsimultaneous to at least a portion of at least one non-water mediacomponent.

In another embodiment, a portion of at least one non-water mediacomponent is added to the fermentation vessel after at least a portionof the XAD-7 resin.

In another embodiment, XAD-7 resin is added in two or more portions.

In another embodiment, at least a portion of the XAD-7 resin is addedsubstantially separate from non-water media components.

In another embodiment, XAD-7 resin is present at the end of the growthphase.

In another embodiment, XAD-7 resin is present while cells aremultiplying.

In another embodiment XAD-7 resin is added during the stationary phase.

In another embodiment, XAD-7 resin is present while cells are producingsalinosporamide A.

Another aspect of the invention relates to a method of producing aproteasome inhibitor, comprising the steps of

preparing a microbial growth medium;

inoculating said medium with Salinispora tropica;

adding a resin to the medium prior to 96 hours after inoculation of themedium.

In one embodiment, the resin is added prior to the onset of thestationary phase of growth.

Another aspect of the invention relates to a method of producingsalinosporamide A, comprising culturing a salinosporamide A producingmicroorganism in the presence of a resin added prior to the fifth day ofthe fermentation.

In one embodiment, the salinosporamide producing microorganism iscultured in a fed-batch mode of operation.

Another aspect of the invention relates to a method of producingsalinosporamide A, comprising culturing a salinosporamide A producingmicroorganism in the presence of a resin selected from the groupconsisting of Amberlite XAD2, Amberlite XAD4, Amberlite XAD7, AmberliteXAD7HP, Amberlite XAD761, Amberlite XAD 761, Amberlite XAD1180, DiaionHP2MG, Diaion HP20, Diaion HP21, Sepabeads SP825, Sepabeads SP850,Sepabeads SP70, Sepabeads SP700, and Sepabeads SP207.

In one embodiment, the resin is added at intervals and these intervalsmay be pre-determined or otherwise based on observable criterion, suchas the extent of cell growth.

In another embodiment, media components are added at intervals duringthe fermentation.

Another aspect of the invention relates to a method of producing acompound of Formula A, wherein R1 is selected from the group consistingof hydrogen and hydroxide, R2 is selected from the group consisting of2-chloroethyl, 2-bromoethyl, ethyl, methyl, and propyl, and R3 isselected from the group consisting of methyl and ethyl, comprising thesteps of growing a culture of S. tropica in the presence of XAD-7 resin.

Another aspect of the invention relates to a method of producing acompound of Formula B, wherein R1 is selected from the group consistingof hydrogen and hydroxide, R2 is selected from the group consisting of2-chloroethyl, 2-bromoethyl, ethyl, methyl, and propyl, and R3 isselected from the group consisting of methyl and ethyl, comprising thesteps of growing a culture of S. tropica in the presence of XAD-7 resin.

Another aspect of the invention relates to a method of producing acompound of Formula C, wherein R2 is selected from the group consistingof 2-chloroethyl and methyl, comprising the steps of growing a cultureof S. tropica in the presence of XAD-7 resin.

One class of marine-derived natural products are Salinosporamide A andanalogs thereof. Salinosporamide A and its analogs have variousbiological activities. For example, the compounds have chemosensitizingactivity, anti-microbial, anti-inflammation, radiosensitizing, andanti-cancer activity. Studies have been conducted that showSalinospormide A and its analogs have proteasome inhibitory activity,effect NF-κB/IκB signaling pathway, and have anti-anthrax activity.Salinosporamide A and several analogs, as well as biological activity ofthe same, are described in U.S. Provisional Patent Applications No.,60/480,270, filed Jun. 20, 2003; 60/566,952, filed Apr. 30, 2004;60/627,461, filed Nov. 12, 2004; 60/633,379, filed Dec. 3, 2004;60/643,922, filed Jan. 13, 2005; 60/658,884, filed Mar. 4, 2005;60/676,533, filed Apr. 29, 2005; 60/567,336, filed Apr. 30, 2004;60/580,838, filed Jun. 18, 2004; 60/591,190, filed Jul. 26, 2004;60/627,462, filed Nov. 12, 2004; 60/644,132, filed Jan. 13, 2005;60/659,385, filed Mar. 4, 2005; 60/791,625, filed Apr. 13, 2006;60/797,553, filed May 3, 2006; and 60/949,147, filed Jul. 11, 2007(entitled “Use of Polyacrylic Resins in Fermentation of MarineActinomycetes”); U.S. patent application Ser. No. 10/871,368, filed Jun.18, 2004; Ser. No. 11/118,260, filed Apr. 29, 2005; Ser. No. 11/412,476,filed Apr. 27, 2006; Ser. No. 11/453,374, filed Jun. 15, 2006; Ser. No.11/517,899, filed Sep. 8, 2006; and Ser. No. 11/697,689, filed Apr. 6,2007 (entitled “Total Synthesis of Salinosporamide A and AnalogsThereof”); and International Patent Applications Nos.,PCT/US2004/019543, filed Jun. 18, 2004; PCT/US2005/044091, filed Dec. 2,2005; PCT/US2005/014846, filed Apr. 29, 2005; and PCT/US2006/016104,filed Apr. 27, 2006; each of which is hereby incorporated by referencein its entirety.

COMPOUNDS

One aspect of the invention relates to producing various useful chemicalcompounds which are made by strains of a S. tropica microorganism. Beloware exemplary compounds:

Various embodiments of compounds having the structure disclosed in theabove Formulae include optically active compounds. One of these isSalinosporamide A, the structure of which is shown below:

Among its properties is its ability to inhibit the production ofproteasomes. Proteasome inhibitors have applicability, among otherthings, in the treatment of cancers. This activity is more fullydescribed in PCT Application Publication No. WO/2006/118973, filed Apr.27, 2006, which is incorporated herein by reference in its entirety.

Other exemplary embodiments of useful compounds produced by S. tropica,which correspond to the formulae described above are identified below bystructure indicating some exemplary stereo-isomers:

Compounds that correspond to the structures of the above formula canexist as different stereo-isomers and should not be limited toindividual forms. For example, Compounds 1 and 7 are two of severalstereo isomers of Formula D. Similarly, Compounds 2 and 8 as well asCompounds 5 and 9 are exemplary stereo isomers of the same Formulastructures. Additional optical forms of the Formulae are alsocontemplated.

Among the useful properties of these compounds are also proteasomeinhibition and the treatment of various diseases including cancers.

Each of these compounds can be used alone or in combination with one ormore other compounds produced by S. tropica, or in combination withother compounds from other sources. Additionally, each of thesecompounds can be chemically modified to change their properties.

The production of Salinosporamide A and analogs thereof can be carriedout by cultivating Salinispora tropica strain NPS021184, ATCC accessionnumber PTA-6685, a natural variant of strain CNB476, in a suitablenutrient medium under conditions described herein, preferably undersubmerged aerobic conditions, until a substantial amount of compoundsare detected in the fermentation; harvesting by extracting the activecomponents from the fermentation broth with a suitable solvent;concentrating the solvent containing the desired components; thensubjecting the concentrated material to chromatographic separation toisolate the compounds from other metabolites also present in thecultivation medium.

In the fermentative production of Salinosporamide A by Salinisporatropica NPS021184, two analogs of Salinosporamide A, NPI-0047, alsoreferred to as Salinosporamide B, and NPI-2065, are co-produced in thefermentation. The separation of NPI-0047 from Salinosporamide A resultsin loss of the recovery yield of Salinosporamide A. Subsequently,removing NPI-2065 from Salinosporamide A leads to further loss inrecovery yield. The structure for each of these compounds is shownbelow:

Accordingly, some embodiments include methods for increasing theproduction of Salinosporamide A while concomitantly reducing productionof NPI-0047 and NPI-2065. In one such embodiment, the addition of Cobaltsalt in the cultivation of Salinispora tropica NPS021184 has been foundto reduce the production of the two Salinosporamide A analogs, NPI-0047and NPI-2065, and increase the production of the desired compoundSalinosporamide A. As previously discussed, decrease in production ofthe two Salinosporamide A analogs leads to decreased loss ofSalinosporamide A during the separation process. This decreased losscombined with the increased production of Salinosporamide A results in ahigher total yield of the desired compound Salinosporamide A when themicroorganism is cultivated in medium containing Cobalt salt as opposedto medium lacking Cobalt salt.

For the growth of Salinispora tropica NPS021184, seed cultures areprepared by inoculating frozen stocks of Salinispora tropica NPS021184into seed medium. Any suitable carbon and nitrogen sources in the seedmedium may be used. In one embodiment, the seed medium contains glucoseat a concentration around 8 g/L and Hy Soy and yeast extract each at aconcentration around 6 g/L. Any of the salt formulations described abovemay be used in the seed formulation. In one embodiment, the saltformulation contains NaCl, MgSO₄, KBr, KCl, CaCO₃, CaCl₂, SrCl₂, H₃BO₃,and NaF salts as described above. In one embodiment, the saltformulation additionally includes CoCl₂.

After an initial incubation in the seed medium, the culture can beinoculated into a second seed medium with the same composition as theoriginal seed medium.

After the second incubation in the seed medium, the culture can beinoculated into a production medium. If production is to be in afermentor culture, a third seed culture may be inoculated beforeinoculation into the production media in the fermentor.

Any suitable carbon and nitrogen sources may be used for the productionmedium. In one embodiment, the production medium contains starch at aconcentration around 10 g/L and concentrations of Hy Soy at about 4 g/Land yeast extract at about 4 g/L. In one embodiment, the productionmedium additionally includes one or more salts (separate from thesupplemental salt formulations described herein). In one embodiment,these salts include CaCO₃, Fe₂(SO₄)₃, and KBr. In one embodiment theCaCO₃ concentration is between 2 mg/L and 50 g/L. In another embodimentthe CaCO₃ concentration is between 100 mg/L and 10 g/L. In the preferredembodiment the CaCO₃ concentration is about 1 g/L. In one embodiment theFe₂(SO₄)₃ concentration is between 0.5 mg/L and 10 g/L. In anotherembodiment the Fe₂(SO₄)₃ concentration is between 5 mg/L and 1 g/L. Inthe preferred embodiment the Fe₂(SO₄)₃ concentration is about 40 mg/L.In one embodiment the KBr concentration is between 0.5 mg/L and 10 g/L.In another embodiment the KBr concentration is between 5 mg/L and 1 g/L.In the preferred embodiment the KBr concentration is about 40 mg/L.

In addition, any of the salt supplement formulations described above maybe added to the production medium. In one embodiment, the saltformulation contains NaCl, KCl, CaCl₂, SrCl₂, H₃BO₃, and NaF salts asdescribed above. In one embodiment, the salt formulation additionallyincludes CoCl₂. In one embodiment, instead of NaCl, the salt formulationcontains Na₂SO₄.

After inoculation into the production medium the production cultures maybe further incubated and harvested at an appropriate time point. In someembodiments, harvesting occurs at various time points to determineoptimal productivity.

Salinosporamide A is a potent proteasome inhibitor, but has only beenproduced at relatively low yields.

Unexpectedly, the present inventors have found that the presence ofparticular resins during the fermentation substantially increases theyield of Salinosporamide A.

The present invention is therefore directed to methods of culturingSalinispora tropica and other proteasome inhibitor producing organismsin the presence of these resins.

The descriptions in the examples provided herein describe work done onthe shake flask scale. However, the invention itself should not beunderstood to be so limited. The invention is adaptable to anyfermentation scale and should be so understood and includes commercial,pilot, laboratory and other scales as well.

Final isolation and purification of the desired compounds can beaccomplished by various methods. For example, the resin can be separatedfrom the culture medium and extracted with solvents such as ethylacetate or other solvent capable of dissolving desired compounds.Alternatively, the resin with the culture medium can be extracted withsolvents, or the culture medium can be extracted with solvents withoutthe resin present, or the resin and medium can be extracted separatelyor sequentially. In some instances, a first product or set of productsmay be isolated from the resin while a second product or set of productsmay be isolated from the medium. Product set one and two may becompletely different or they may both contain at least some of the sameproducts.

Further processing of the products collected from the culture mediumand/or resin can be performed to increase purity, separate one productfrom another, or chemically modify the one or more of the compound. Anyappropriate processing technique can be used including but not limitedto chromatography, crystallization, adsorption, absorption, evaporation,precipitation, distillation, extraction, and electrophoresis.

The fermentation medium described herein is exemplary. Any medium thatproduces growth of the organism and allows the practice of the inventioncan be used. Likewise, the fermentation conditions described areexemplary and can be modified without straying from the practice of theinvention.

One embodiment of a fermentation medium which can be used to grow theorganism includes a carbon source and a nitrogen source. Exemplarycarbon sources include starch, maltodextrin, glucose, sucrose, fructose,glycerol, monosaccharides, disaccharides, oligosaccharides,polysaccharides, lactic acid, lactose, maltose and materials thatcontain any of these or other carbon containing compounds that aredigestible by the organism alone or in combination. Exemplary nitrogensources include, but are not limited to, proteins, amino acids,monosodium glutamate, ammonia, ammonium hydroxide, urea, corn steepliquor, corn steep solids, yeast-derived materials, and soy-derivedproducts. Some nitrogen sources may also serve as a carbon source andsome carbon sources may also serve as a nitrogen source.

An embodiment of the medium includes a carbon source, a nitrogen source,and inorganic salts. Some or all of the salts can be included in thewater that is used for the fermentation or the carbon or nitrogensource. Examples of salt that is a part of the water would be the saltsthat become a part of the medium through the use of corn steep liquor ortap water or other non-distilled or non fully deionized water. Inanother embodiment, salts are added in addition to those supplied by thewater, carbon or nitrogen source. These added salts may include some,all, or none of those species supplied by the other sources. In anotherembodiment, the salt combinations are those that are disclosed in U.S.Provisional Application No. 60/846,774, filed Sep. 22, 2006, the entiredisclosure of which is incorporated by reference herein. Anotherembodiment utilizes the material, INSTANT OCEAN® (Aquarium Systems,Wickliffe, Ohio) as the salt source or one of the salt sources. Otherembodiments utilize the salt mixtures of Tables D-1 and D-2 as the salt

TABLE D-1 Salt component per liter of DI H₂O NaCl 24.04 g KCl 686.8 mgCaCl₂•2H₂O 429.3 mg SrCl₂ 15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52μg

TABLE D-2 Salt component per liter of DI H₂O NaCl 24.04 g MgSO₄•7H₂O4.29 g KBr 85.9 mg KCl 686.8 mg CaCO₃ 429.3 mg CaCl₂•2H₂O 429.3 mg SrCl₂15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg NiSO₄ 57.7 μg CoCl₂•6H₂O 207.9 μg

Resins

Solid resins may be added to the fermentation. Adding an appropriateresin or combination of resins to the fermentation may increase yield ofparticular compounds produced by the microorganism. In one embodiment, asolid resin is added to the fermentation prior to inoculating thefermenter with the desired microorganism. In another, the resin is addedafter inoculating the fermenter with the desired microorganism. Inanother embodiment, the resin is added to an earlier stage of thefermentation. In other embodiments, resin is present in a fermenter thatis operated in a fed-batch or semicontinuous manner. In anotherembodiment, the resin is added prior to the onset of the stationaryphase of the growth cycle of the organism. In another embodiment, theresin is mixed with medium components prior to addition to thefermentation vessel. In another embodiment the resin is added at thesame time as at least one medium component. In another embodiment atleast one medium component is added after the resin. In anotherembodiment at least one medium component is added prior to adding theresin. In another embodiment, the resin is added approximately at thepoint where the stationary phase of growth starts. In anotherembodiment, the resin is added during the stationary phase of growth.

Generally, resins adsorb chemical species to their surface. The degreeof adsorption is governed by a number of factors including, for example,the concentration of the compound, the polarity of the compound, thefunctional groups present on the compound, the composition of the resin,the chemical environment, temperature, the presence or absence of othercompounds that will compete with the targeted compound for adsorption tothe resin, and the pore structure of the resin such as pore diameter andpore volume. When used in conjunction with living cells, the resin mayadsorb one or more nutrients, thus depriving the cell of a materialneeded for growth or production of metabolites. The resin may alsoimmobilize extracellular enzymes, suppress or inactivate enzymes andeven immobilize the cells.

Exemplary resins are described in Table 2 according to theircomposition, pore size and porosity. These and other resins have beenused in fermentations. For example, Lam, et al. showed an increase intiter of dynemicins from Micromonospora chersina with the addition of 1%of Diaion HP-20, but completely stopped production of this compound with4% of this resin. K. S. Lam, et al., Effect of Neutral Resins on theProduction of Dynemicins by Micromonospora chersina, Journal ofIndustrial Microbiology (1995) 15, 453-456.

TABLE 2 Resins and physical characteristics Surface Pore Particle arearadius Porosity size Density Resin Structure (m²/g) (Å) (mL/g) (mm)(g/mL) XAD-2 SDVB* 330 45 0.65 0.25-0.84 1.08 XAD-4 SDVB* 725 20 0.980.25-0.84 1.02 XAD-7 Acrylic 450 45 1.14 0.25-0.84 1.24 XAD-16 SDVB* 80050 1.82 0.25-0.84 1.08 IRA-67 Acrylic- 0.3-1.2 DVB** IRC-50 Acrylic-0.3-1.2 DVB** IRP-64 Methacrylic- 0.04-0.15 DVB** HP-20 Aromatic511 >200 1.18 >0.25 1.01 HP-2MG Methacrylic 473 200 1.15 >0.25 1.09SP-207 Modified 627 110 0.79 >0.30 1.18 Aromatic SP-850 Aromatic 995 381.2 >0.25 *SDVB = Styrene-divinylbenzene; **DVB = Divinylbenzene

These resins are made by Rohm & Haas except the “HP” and “SP” resins,which are made by Mitsubishi Chemical Corp.

For the present class of organisms and class of products, it hassurprisingly been found that certain resins present during thefermentation result in a substantial increase in the production ofuseful products. As is demonstrated in the following examples, someaspects of the present invention relate to the type of resin that isused, the time of when the resin is presented to the fermentation, andthe amount of resin that is used.

The following non-limiting examples are meant to describe the preferredembodiments of the methods. Variations in the details of the particularmethods employed and in the precise chemical compositions obtained willundoubtedly be appreciated by those of skill in the art. Throughoutthese examples, starch, peptone, and yeast extract were used in variousfermentation. The particular products used were starch, USB catalog no.21695; peptone, USB catalog no. 20048; and yeast extract, USB catalogno. 23547. However, it is envisioned that other carbon and nitrogensources can be used as well.

Example 1 Examination of the Effect of Cobalt in Production Media on theProduction of Salinosporamide A. NPI-0047 and NPI-2065

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into seed medium.

The seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then supplemented with INSTANT OCEAN® commercial salt formulation ata concentration of 30 g/L. No CoCl₂ was added to the seed medium.

The first seed cultures (either 10 mL medium in a 50-mL tube or 100 mLmedium in a 500-mL Erlenmeyer flask) were incubated for 3 to 4 daysbefore inoculating into the second seed media with the same compositionas the first seed cultures. The second seed cultures (100 mL medium in a500-mL Erlenmeyer flask) were incubated for two days before inoculatinginto the production medium (100 mL medium in a 500-mL Erlenmeyer flask).

Per liter of DI H₂O, the production medium consisted of: 10 g Starch, 4g Hy So, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, and 100 mg KBr.The production medium was then supplemented with a chemically definedsalt formulation, with and without various concentrations of CoCl₂. Thechemically defined salt formulation included:

Salt component per liter of DI H₂O NaCl 24 g KCl 0.69 g CaCl₂•2H₂O 0.43g SrCl₂ 15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg

Between 24 and 48 hours of inoculation into the production cultures,sterile XAD-7 resin slurry was added to the production cultures in afinal concentration of 20 g/L. The production cultures were then furtherincubated and harvested at various time points to determineproductivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on a shaker for one hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

Effect of Cobalt Concentration on the Production of Salinosporamide A,NPI-0047, and NPI-2065 in a Shake Flask Culture

CoCl₂.6H₂O was added to the production medium in concentrations rangingfrom 2.6 μg/L to 10.4 mg/L. No CoCl₂.6H₂O was added to the productionmedium in control samples. The production of Salinosporamide A,NPI-0047, and NPI-2065 was measured to determine the effect of thepresence of cobalt as well as the effect of varying cobaltconcentrations in the production media.

Results:

TABLE 1 The Effect of a Wide Range of Cobalt Concentrations in theProduction Media on the Production of Salinosporamide A, NPI-0047, andNPI 2065 Maximum Titer (mg/L) % Increase (+) or % decrease (−)CoCl₂·6H₂O Salinosporamide NP1- NP1- Salinosporamide NPI- NPI-Concentration A 0047 2065 A 0047 2065 No addition 248.0 10.6 7.7 — — —104 μg/L 251.7 3.6 6.1 1.5 −65.9 −21.0 1.04 mg/L 253.4 3.5 6.8 2.2 −67.4−12.2 10.4 mg/L 244.6 3.1 5.3 −1.4 −71.3 −30.8

The production yield of Salinosporamide A only changed negligibly withincreasing concentrations of CoCl₂.6H₂O, up to 10.4 mg/L, in theproduction medium. In contrast, the production of NPI-0047 decreaseddramatically by 66% when 104 μg/L CoCl₂.6H₂O was added to the productionmedium. An increase in the concentration of CoCl₂.6H₂O by 100-fold to10.4 mg/L did not yield any further significant decrease in NPI-0047production. The production of NPI-2065 was inhibited by 30.8% in thepresence of CoCl₂.6H₂O at a concentration of 10.4 mg/L in the productionmedium as compared to production media lacking CoCl₂.6H₂O. The presenceof CoCl₂.6H₂O in the production medium also inhibited the production oforange pigment in the fermentation.

Determination of the Optimal Cobalt Concentration in the ProductionMedium to Inhibit the Production of NPI-0047

The conditions described above were left unchanged except that aCoCl₂.6H₂O concentrations ranging from 2.6 μg/L to 416 μg/L was added tothe production media to determine the optimal cobalt concentration inproduction media to inhibit the production of NPI-0047. No CoCl₂.6H₂Owas added to control samples.

The production of Salinosporamide A, NPI-0047, and NPI-2065 was measuredto determine the effect of the presence of cobalt as well as the effectof varying cobalt concentrations in the production media. Of particularinterest was the effect of cobalt concentration on the production ofNPI-0047.

Results:

TABLE 2 The Effect of Cobalt in the Production Media on the Productionof Salinosporamide A, NPI-0047, and NPI-2065 CoCl₂·6H₂O Max Titers(mg/L) % Increase (+) or % decrease (−) Concentration Saiinosporamide ANPI-0047 NPI-2065 SALINOSPORAMIDE A NPI-0047 NPI-2065 Control (No 272.310.2 6.6 — — — addition) 2.6 μg/L 275.3 8.3 6.1 +1.1 −18.6 −7.6 5.2 μg/L247.9 6.0 6.5 −8.9 −41.2 −1.5 6.5 μg/L 275.7 6.3 6.2 +1.2 −38.2 −6.1  13μg/L 271.3 4.9 6.5 −0.3 −52.0 −1.5  26 μg/L 261.4 3.7 6.8 −4.0 −63.7+3.0  52 μg/L 259.9 3.1 6.3 −4.5 −69.6 −4.6 104 μg/L 264.2 3.5 6.5 −3.0−65.7 −1.5 208 μg/L 272.8 3.1 6.2 +0.2 −69.6 −6.1 416 μg/L 249.1 2.7 5.6−8.5 −73.5 −15.2

An increasing concentration of cobalt in the production mediumcorrelated with a decreasing production of NPI-0047. The inhibitioneffect of cobalt on the production of NPI-0047 leveled off at 52 μg/Lcobalt concentration. At 52 μg/L cobalt concentration, the production ofNPI-0047 was reduced by 69.6%. The production of NPI-2065 was alsoreduced in cobalt-supplemented conditions with the maximum inhibitioneffect (15% inhibition) observed at 416 μg/L, the highest concentrationcobalt tested. The production of Salinosporamide A was not affected bythe presence of cobalt.

Example 2 The Effect of Cobalt in Seed Media on the Production ofSalinosporamide A, NPI-0047 and NPI-2065

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into seed medium.

The seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then supplemented with a chemically defined salt formulation. Thechemically defined salt formulation included:

Salt component per liter of DI H₂O NaCl 24 g MgSO₄•7H₂O 4.29 g KBr 85.9mg KCl 0.69 g CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15.5 mg H₃BO₃ 21.5 mgNaF 2.6 mg

The seed medium was also supplemented with CoCl₂.6H₂O at concentrationsranging from 2.6 μg/L to 416 μg/L. No CoCl₂.6H₂O was added to controlsamples.

The first seed cultures (either 10 mL medium in a 50-mL tube or 100 mlmedium in a 500-ml Erlenmeyer flask) were incubated for 3 to 4 daysbefore inoculating into the second seed media with the same compositionas the first seed cultures. The second seed cultures (100 mL medium in a500-mL Erlenmeyer flask) were incubated for two days before inoculatinginto the production medium (100 mL medium in a 500-mL Erlenmeyer flask).

Per liter of DI H₂O, the production medium consisted of: 10 g Starch, 4g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, and 100 mg KBr.The production medium was then supplemented with a chemically definedsalt formulation. The chemically defined salt formulation consisted of:

Salt component per liter of DI H₂O NaCl 24 g KCl 0.69 g CaCl₂•2H₂O 0.43g SrCl₂ 15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg

Between 24 and 48 hours of inoculation into the production cultures,sterile XAD-7 resin slurry was added to the production cultures in afinal concentration of 20 g/L. The production cultures were then furtherincubated and harvested at various time points to determineproductivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

Determination of the Cobalt Concentration in the Seed Medium Required toOptimize Production Salinosporamide A and Inhibition of NPI-0047 andNPI-2065

The seed medium was supplemented with CoCl₂.6H₂O at concentrationsranging from 2.6 μg/L to 416 μg/L. No CoCl₂.6H₂O was added in controlsamples. CoCl₂.6H₂O was added to production cultures at a concentrationof 52 μg/L.

The production of Salinosporamide A, NPI-0047, and NPI-2065 was measuredto determine the effect of the presence of cobalt as well as the effectof varying cobalt concentrations in the seed media. The optimal cobaltconcentration in the seed medium was determined by analyzing theproduction of Salinosporamide A, as well as the percent NPI-0047 andNPI-2065 produced relative to the Salinosporamide A produced.

Results

TABLE 3 Determination of the optimal cobalt concentration in the seedmedium relative to Sal CoCl₂·6H₂O Max Avg Titer (mg/L) A (%) %increase/decrease relative % change Concentration NPI- NPI- NPI- NPI-NPI- NPI- NPI- NPI- (Seed) Sal A 0047 2065 0047 2065 Sal A 0047 20650047 2065  0 μg/L 291.8 3.3 6.5 1.1 2.2 0.0 0.0 0.0 0.0 0.0 2.6 μg/L296.8 3.5 6.6 1.2 2.2 1.7 5.9 1.7 4.2 0.0 5.2 μg/L 306.0 3.4 6.8 1.1 2.24.7 2.0 4.2 −2.7 −0.5  13 μg/L 299.4 2.8 6.9 0.9 2.3 2.5 −17.2 5.9 −20.23.5  26 μg/L 366.2 3.2 8.4 0.9 2.3 20.3 −2.3 23.1 −28.4 3.5  52 μg/L326.6 2.7 7.9 0.8 2.4 10.7 −21.6 17.7 −36.1 7.9 104 μg/L 347.0 3.0 7.70.9 2.2 15.9 −9.2 15.5 −29.9 −0.5 208 μg/L 368.5 2.9 8.1 0.8 2.2 20.8−11.9 19.7 −41.3 −1.4 416 μg/L 321.2 3.0 6.9 0.9 2.2 9.2 −10.4 6.2 −21.5−3 Note: “Sal A” refers to Salinosporamide AThe optimal concentration of CoCl2.6H2O in the seed medium wasdetermined to be 208 g/L. This was based on the 20.8% improvement inSalinosporamide A production, the 41.3% relative decrease in NPI-0047production, and the 1.4% relative decrease in NPI-2065 production.

Determination of the Effect of Cobalt Concentration in the Seed Mediumon the Production of Salinosporamide A, NPI-0047, and NPI-2065

The seed medium was supplemented with CoCl₂.6H₂O at a concentration of208 μg/L. No CoCl₂.6H₂O was added to the seed medium in control samples.CoCl₂.6H₂O was added to production medium at a concentration of 52 μg/L.No CoCl₂.6H₂O was added to the production medium in control samples. Theproduction of Salinosporamide A, NPI-0047, and NPI-2065 was measured todetermine the effect of the presence of cobalt in the seed medium andproduction medium.

Results:

TABLE 4 Effect of cobalt concentration in the seed medium on theproduction of Salinosporamide A, NPI-0047, and NPI-2065 CoCl₂·6H₂OCoCl₂·6H₂O Average Max Titer (mg/L) % Increase (+) or % decrease (−) inseed in production SALINOSPORAMIDE NPI- NPI- SALINOSPORAMIDE NPI- NPI-medium medium A 0047 2065 A 0047 2065 0 0 227.6 12.7 7.8 — — — 0 52 μg/L250.6 3.6 4.9 +10.1 −71.7 −37.2 208 μg/L 52 μg/L 304.7 2.8 4.3 +33.9−78.0 −44.9 (Average of three experiments)

Addition of cobalt to both the seed and production media increased theproduction of Salinosporamide A by 33.9% as compared to seed andproduction media without cobalt. Additionally, the production ofNPI-0047 was reduced by 78.0% and production of NPI-2065 was reduced by44.9%.

As compared to conditions where cobalt was added only to the productionmedium, the presence of cobalt in the seed medium and production mediumincreased the production of Salinosporamide A by 21.6%, reduced theproduction of NPI-0047 by 22.2% and reduced the production of NPI-2065by 12.2%. This result demonstrates the benefit of adding cobalt to bothseed and production media.

Example 3 Effect of Cobalt on the Production of Salinosporamide A.NPI-0047 and NPI-2065 in Fermentor Culture

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into seed medium. For 10 mL seed medium in a 50-mLculture tube, one frozen stock (˜1.5 mL) was used for inoculation. For100 mL seed medium in a 500-mL Erlenmeyer flask, two frozen stocks (˜3mL total) were used for inoculation.

The seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then further supplemented with either INSTANT OCEAN® (AquariumSystems, Inc., Wickliffe, Ohio) commercial salt formulation or achemically defined salt formulation. This chemically defined formulationconsisted of:

Salt component per liter of DI H₂O NaCl 24 g MgSO₄•7H₂O 4.29 g KBr 85.9mg KCl 0.69 g CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15.5 mg H₃BO₃ 21.5 mgNaF 2.6 mg

The seed medium was also supplemented with CoCl₂.6H₂O at a concentrationof 208 μg/L. No CoCl₂.6H₂O was added to the seed medium in controlsamples.

The first seed cultures (either 10 mL medium in a 50-mL tube or 100 mLmedium in a 500-mL Erlenmeyer flask) were incubated at 250 rpm and 28°C. for 3 to 4 days before inoculating (5% inoculum) into second seedmedium with the same salt composition as used for the first seedcultures. After incubating at 250 rpm and 28° C. for 2 days, the secondseed cultures (5% inoculum) were inoculated into an additional thirdseed culture (400 mL medium in 2.8 L Fembach flask) with the samemedium, salt composition and incubation time as the second seedcultures, before inoculating into production media in the fermentor (26L medium in a 42 L B. Braun Biostat-C fermentor).

Per liter of DI H₂O, the production medium consisted of:

 10 g Starch   4 g Hy Soy   4 g Yeast Extract   1 g CaCO₃  40 mgFe₂(SO₄)₃ 100 mg KBrThe production medium was then supplemented with either INSTANT OCEAN®commercial salt formulation or a chemically defined salt formulationbased on the salt supplement used in the seed stages. The chemicallydefined salt formulation consisted of:

Salt component per liter of DI H₂O NaCl 24 g KCl 0.69 g mg CaCl₂•2H₂O0.43 g mg SrCl₂ 15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg

The production medium for fermentation was also supplemented withCoCl₂.6H₂O at a concentration of 52 μg/L. No CoCl₂.6H₂O was added to theproduction medium in control samples.

Between 24 and 48 hours of inoculation into the production cultures,sterile XAD-7 resin slurry was added to the production cultures in afinal concentration of 20 g/L. The production cultures were then furtherincubated and harvested at various time points to determineproductivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture.

The mixture was shaken for 1 hr. An aliquot of the extract (1 mL) wasremoved and dried under a stream of nitrogen. The dried extract wasstored at −20° C. freezer before HPLC analysis. The dried extract wasresuspended in 320 μL DMSO and injected into HPLC using the 00TF14method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

Effect of Cobalt on the Production of Salinosporamide A. NPI-0047, andNPI-2065 Incubated in Seed Medium Supplemented with INSTANT OCEAN®Commercial Salt Formulation and Fermented in Production Medium AlsoSupplemented with INSTANT OCEAN® Commercial Salt Formulation.

The seed medium was supplemented with INSTANT OCEAN® salt formulation ata concentration of 30 g/L. No CoCl₂.6H₂O was added to the seed medium.The production medium was supplemented with INSTANT OCEAN® saltformulation at a concentration of 30 g/L. No CoCl₂.6H₂O was added to theproduction medium.

The production of Salinosporamide A, NPI-0047, and NPI-2065 wasmeasured. The results are presented below in Table 5.

TABLE 5 Production of Salinosporamide A, NPI-0047, and NPI-2065incubated in seed medium and fermented in production medium eachsupplemented with INSTANT OCEAN ® commercial salt formulation in theabsence of cobalt %NPI- %NPI- SALINO- 0047 as 2065 as SPORA- NPI- NPI-SALINO- SALINO- MIDE 0047 2065 SPORAMIDE SPORAMIDE Batch # A (mg/L)(mg/L) (mg/L) A A C27 260 9.8 9.6 3.8 3.7 C29 250 13.8 11.0 5.5 4.4 C30260 8.3 9.1 3.2 3.5 Average 257 10.6 9.9 4.1 3.9

To test the effect of cobalt, seed medium was supplemented with INSTANTOCEAN® salt formulation at a concentration of 30 g/L. CoCl₂.6H₂O wasadded to the seed medium at a concentration of 208 μg/L. The productionmedium was supplemented with INSTANT OCEAN® salt formulation at aconcentration of 30 g/L. CoCl₂.6H₂O was added to the production mediumat a concentration of 52 μg/L. The production of Salinosporamide A,NPI-0047, and NPI-2065 was measured to determine the effect of cobaltadded to the seed medium and production medium. The results arepresented below in Table 6.

TABLE 6 Production of Salinosporamide A, NPI-0047, and NPI-2065incubated in seed medium and fermented in production medium eachsupplemented with INSTANT OCEAN ® commercial salt formulation in thepresence of cobalt RATIO OF RATIO OF SALINO- SALINO- SALINO- SPORA- NPI-NPI- SPORAMIDE SPORAMIDE MIDE 0047 2065 A TO A TO Batch # A (mg/L)(mg/L) (mg/L) NPI-0047, % NPI-2065, % C49 340 6.5 9.2 1.9 2.7 C50 2955.0 7.7 1.7 2.6 C53 265 5.0 5.8 1.9 2.2 Average 300 5.5 7.6 1.8 2.5

Results:

With the addition of cobalt in both the seed and production media usingINSTANT OCEAN® salt formulation, on average the production ofSalinosporamide A increased by 14.3%, the production of NPI-0047decreased by 48.1%, and production of NPI-2065 decreased by 23.2%.

Effect of Salt Formulations on the Production of Salinosporamide A.NPI-0047, and NPI-2065.

The seed medium was supplemented with the chemically defined saltformulation. The seed medium was also supplemented with CoCl₂.6H₂O at aconcentration of 208 μg/L.

The production medium was supplemented with the chemically defined saltformulation. The production medium was supplemented with CoCl₂.6H₂O at aconcentration of 52 μg/L.

The production of Salinosporamide A, NPI-0047, and NPI-2065 was measuredto compare production by cultures grown in chemically defined saltsupplemented seed medium and chemically defined salt supplementedproduction medium with production by cultures grown in seed andproduction media supplemented with INSTANT OCEAN® commercial saltformulation presented in tables 5 and 6 above.

TABLE 7 Production of_Salinosporamide A, NPI-0047, and NPI-2065 producedby cultures grown in chemically defined salt supplemented seed mediumand chemically defined salt supplemented production medium in thepresence of cobalt. RATIO OF RATIO OF SALINO- SALINO- SALINO- SPORA-NPI- NPI- SPORAMIDE SPORAMIDE MIDE 0047 2065 A TO A TO Batch # A (mg/L)(mg/L) (mg/L) NPI-0047, % NPI-2065, % C39 280 3.4 7.0 1.2 2.5 C42 2672.4 6.9 0.9 2.6 Average 274 2.9 7.0 1.1 2.6

Results

TABLE 8 Comparison of Production of Salinosporamide A, NPI-0047, andNPI-2065 in seed and production media supplemented with or withoutcobalt in a 42 L fermentor NPI- NPI- RATIO OF RATIO OF SAL A 0047 2065SAL A TO SAL A TO Media (mg/L) (mg/L) (mg/L) NPI-0047, % NPI-2065, %Seed: Instant Ocean no cobalt 257 10.6 9.9 4.1 3.9 Production: InstantOcean no cobalt Seed: Instant Ocean + 208 μg/L 300 5.5 7.6 1.8 2.5CoCl₂•6H₂O Production: Instant Ocean + 52 μg/L CoCl₂•6H₂O Seed:Chemically Defined Salt + 274 2.9 7.0 1.1 2.6 208 μg/L CoCl₂•6H₂OProduction: Chemically Defined Salt + 52 μg/L CoCl₂•6H₂O SAL A isSalinosporamide A

By replacing the INSTANT OCEAN® commercial salt formulation with achemically defined salt formulation in the seed medium and a chemicallydefined salt formulation with cobalt chloride in the production medium,the production of NPI-0047 was reduced by 47.3% and the production ofNPI-2065 was reduced by 7.9%. The production of Salinosporamide Adecreased by 8.7%.

Example 4 The Effect of Non-Saline, “Low Chloride”, Salt Formulation onthe Production of Salinosporamide A, NPI-0047, and NPI-2065

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into seed medium.

The seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then supplemented with a chemically defined salt formulation. Thechemically formulation consisted of:

Salt component per liter of DI H₂O NaCl 29 g MgSO₄•7H₂O 4.29 g KBr 85.9mg KCl 0.69 g CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15.5 mg H₃BO₃ 21.5 mgNaF 2.6 mg

The seed medium was supplemented with CoCl₂.6H₂O at a concentration of208 μg/L. No CoCl₂.6H₂O was added to the seed medium in control samples.

The first seed cultures (either 10 mL medium in a 50-ml tube or 100 mLmedium in a 500-mL Erlenmeyer flask) were incubated for 3 to 4 daysbefore inoculating into the second seed media with the same compositionas the first seed cultures. The second seed cultures (100 mL medium in a500-mL Erlenmeyer flask) were incubated for two days before inoculatinginto production medium (100 mL medium in a 500-mL Erlenmeyer flask).

Per liter of DI H₂O, the production medium consisted of: 10 g Starch, 4g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, and 100 mg KBr.The production medium was then either supplemented with INSTANT OCEAN®commercial salt formulation or supplemented with a chemically definedsalt formulation. This chemically defined salt formulation consisted of:

Salt component per liter of DI H₂O KCl 0.69 g CaCl₂•2H₂O 0.43 g SrCl₂15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg

The production medium with the chemically defined salt formulation wasthen supplemented with either NaCl at a concentration of 24 g/L orNa₂SO₄ at a concentration of 15 g/L. Production medium containing thechemically defined salt formulation and NaCl was either supplementedwith CoCl₂.6H₂O at a concentration of 52 μg/L or not at all. Productionmedium containing the chemically defined salt formulation and Na₂SO₄ wassupplemented at a concentration of 52 μg/L.

Between 24 and 48 hours after inoculation, sterile XAD-7 resin slurrywas added to the production cultures in a final concentration of 20 g/L.The production cultures were then further incubated and harvested atvarious time points to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken for 1 hr. An aliquot of theextract (1 mL) was removed and dried under a stream of nitrogen. Thedried extract was stored at −20° C. freezer before HPLC analysis. Thedried extract was resuspended in 320 JAL DMSO and injected into HPLCusing the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min for 15min with the detector wavelength set at 210 nm and column temperature35° C.

Results:

TABLE 9 Production of Salinosporamide A, NPI-0047, and NPI-2065 inproduction media with various salt compositions Sal A NPI-0047 NPI-2065Media (mg/L) (mg/L) (mg/L) INSTANT OCEAN ® 228 12.7 7.8 ChemicallyDefined Salt Formulation + 248 10.6 7.7 NaCl Chemically Defined SaltFormulation +  279* 3.2* 5.2* NaCl + 52 μg/L CoCl₂•6H₂O ChemicallyDefined Salt Formulation + 217 7.3 3.4 Na₂SO₄ + 52 μg/L CoCl₂•6H₂O(Medium Lo.Maz) *Average of two experiments

Particular ions present in the low chloride, Na₂SO₄ based Medium Lo.Mazwere analyzed by ICP-MS analysis and are listed in the table below:

TABLE 10 ICP-MS Analysis of Ions of Interest in Lo. Maz Medium, mMMedium [Na] [Cl] [K] [Mg] [Co] [S] Lo.Maz 222 17 16 0.49 3.1 x 10⁻⁴ 124

The chemically defined salt formulation with Na₂SO₄ significantlyreduced chloride content (17 mM) and reduced sodium content (222 mM).This low chloride, non-saline salt formulation is significantly lesscorrosive to fermentor than the regular saline salt formulation. Asevidenced by Table 9, the production of Salinosporamide A, NPI-0047, andNPI-2065 was only minimally reduced as compared to the chemicallydefined salt formulation with NaCl.

Example 5 Growth of Salinispora tropica NPS021184 and Production ofSalinosporamide A in a Non-Saline, Low Sodium, Production MediumContaining Trace Amount of Sodium Ion (Calculated Value:10.7 mM; ICP-MSAnalysis: 16 mM)

Seed Cultures were prepared by inoculating a frozen stock of Salinisporatropica NPS021184 into seed medium.

The seed medium consisted of the following per liter of DI water: 10 gStarch, 4 g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, and 40 mg Fe₂(SO₄)₃.The seed medium was then supplemented with the chemically defined saltformulation shown below. This supplemented medium is referred to asLo.BNA.

Salt component Per liter of DI H₂O Na₂SO₄ 15 g MgSO₄•7H₂O 4.29 g KBr85.9 mg KCl 0.69 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6mg CoCl₂•6H₂O 208 μg

The first seed culture (10 mL medium) in a 50-mL culture tube wasincubated for 72 hours before inoculating 5 mL of the first seed cultureinto the second seed medium with the same composition as the first seedmedium. The second seed culture (100 mL medium in a 500-mL Erlenmeyerflask) was incubated for 48 hours before inoculating 5 mL of the secondseed culture into the production medium (100 mL in a 500-mL Erlenmeyerflask) supplemented with trace amount of sodium ion (0.06 mM from NaF).

Per liter of DI H₂O, the production medium consisted of: 10 g Starch, 4g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, and 100 mg KBr.The production medium was then supplemented with the chemically definedsalt formulation shown below. This supplemented media is referred to asSHY.KcMC.

Salt component Per liter of DI H₂O KCl 30 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52 μg

The production medium was supplemented with 0.06 mM sodium ion derivedfrom NaF (2.6 mg/L). The major source of the sodium ion in theproduction culture was derived from the 5% seed inoculum (5 mL seedculture into 100 mL production medium), which contained 211 mM sodiumion from 15 g/L sodium sulfate. Therefore, the 5% seed inoculum supplied10.6 mM sodium ion to the production culture. The calculated amount ofsodium ion present in the production medium was 10.7 mM (0.06 mM+10.6mM). The amount of particular ions in the seed medium, production mediumand production after inoculation of 5% of seed medium by ICP-MS analysisare listed in the following table.

TABLE 11 ICP-MS analysis of key ion concentration in seed medium Lo.BNA,Production medium SHY.KcMC and Medium SHY.KcMC with 5% Lo.BNA [Na] [Cl][K] [Mg] [Co] [S] [Ca] [Fe] Seed medium 215 17 13 19 1.2 x 10⁻³ 143 3.80.17 (Lo.BNA) Production 11 477 468 0.75 3.7 x 10⁻⁴ 1.7 4.1 0.12 medium(SHY.KcMC) Production 16 453 411 1.6 3.9 x 10⁻⁴ 8.8 3.8 0.12 mediumafter inoculation of 5% seed medium

About 24 hour after inoculation, sterile XAD-7 resin slurry was added tothe production cultures in a final concentration of 20 g/L. Theproduction culture was then further incubated and harvested at varioustime points to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and an isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min for 15min with the detector wavelength set at 210 nm and column temperature35° C.

TABLE 12 Production of Salinosporamide A, NP1-0047 and NP1-2065 bySalinispora tropica NPS021184 grown in production medium containing~10.7 mM sodium ion (calculated value) and 16 mM sodium ion determinedby ICP-MS analysis. Titer (mg/L) Culture Age (days) Salinosporamide ANPI-0047 NPI-2065 3 109 0.7 0.9 4 187 1.1 2.5 5 226 0.3 3.5 6 239 0.54.0

While the amount of sodium ion known to be present the production mediumSHY.KcMC is 0.06 mM based on the addition of 2.6 mg/L NaF, ICP-MSanalysis showed that the actual sodium ion content in production mediumSHY.KcMC is 11 mM. The additional sodium present in the productionmedium SHY.KcMC is derived from the other medium components such as HySoy and yeast extract (see Table 20 of Example 9). By the same token,the calculated amount of sodium present in the production medium afterthe inoculation of seed medium containing high sodium content is 10.7 mMwhile the actual sodium content determined by ICP-MS analysis is 16 mM.

Salinispora tropica NPS21184 can be grown in production mediumcontaining low sodium ion concentration at 16 mM, about 3% of the sodiumconcentration present in sea water (487 mM sodium, see Table 20 ofExample 9). In this low sodium production medium, Salinispora tropicaNPS21184 produced 239 mg/L of Salinosporamide A.

Example 6 Growth of Salinispora tropica NPS021184 and Production ofSalinosporamide A in Non-Saline, Low Sodium, Seed and Production MediaContaining Trace Amount of Sodium Ion (Calculated Value: 0.06 mM: ICP-MSAnalysis: 11 mM)

The first seed culture was prepared by inoculating two frozen stocks ofSalinispora tropica NPS021184 into first seed medium containing INSTANTOCEAN® commercial salt formulation to obtain good cell growth. The firstseed culture was centrifuged and washed with Wash Media to remove thesodium ion present in the first seed culture before inoculating into thefirst of two additional seed stages. The medium used for these last twoseed stages contained only trace amounts of sodium ion (calculated valueof sodium based on the added NaF: 0.06 mM; estimated to be around 5.7 mMbased on analysis of a similar medium, A1.Ks4C, in Example 7). The thirdseed culture was then inoculated into the production medium SHY.KcMCcontaining only trace amounts of sodium ion (calculated value of sodiumderived from the added NaF: 0.06 mM; ICP-MS analysis: 11 mM). Thisprocess significantly reduced the carryover of sodium ion from the seedcultures to the production culture.

The first seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then supplemented with 30 g/L INSTANT OCEAN® commercial saltformulation.

The first seed culture (100 ml medium containing ˜424 mM sodium ion fromInstant Ocean by ICP-MS analysis, Table 20 in Example 9) in a 500-mLErlenmeyer flask was incubated at 28° C. and 250 rpm for 72 hours. 10 mLof the first seed culture was transferred to a sterile 15-mL centrifugetube and centrifuged at 3,000 rpm for 15 min. The packed cell volumeobserved was 3%. The supernatant was decanted and 10 mL of Wash Medium(10 g/L starch, 2 g/L peptone, 4 g/L yeast extract, and 30 g/L KCl) wasadded to the centrifuge tube. The centrifuge tube was mixed and thencentrifuged at 3000 rpm for 15 min. The supernatant was decanted and thecells were centrifuged, suspended in Wash Media, and centrifuged againas described above. After the final washing, 10 mL of the second seedmedium (containing 0.06 mM sodium ion from NaF) was added to thecentrifuge tube. The second seed medium consisted of 10 g/L starch (USB,catalog #21695), 2 g/L peptone (USB, catalog #20048), 4 g/L yeastextract (USB, catalog #23547) and the chemically defined saltformulation shown below. This medium was called A1.Kc4C

Salt component Per liter of DI H₂O KCl 30 g MgSO₄•7H₂O 4.29 g KBr 85.9mg CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mgCoCl₂•6H₂O 208 μg

The sodium content in the second seed medium should be around 5.7 mMbased on the ICP-MS analysis of a similar medium, A1.Ks4C (Example 7, 30g/L KCl instead of 30 g/L K₂SO₄), and the sodium content present in A1media (5.2 mM sodium, Table 20 of Example 9). The centrifuge tube wasgently vortexed to mix the cell suspension and 5 mL of the washed cellswas inoculated to the second seed culture containing 100 ml mediumhaving the same composition as the second seed medium in a 500-mLErlenmeyer flask. The sodium concentration in the washed cell inoculumhad been reduced down to a calculated value of ˜0.07 mM based on 424 mMsodium from the INSTANT OCEAN® supplement and 3% packed cell volume.This value is similar to the second seed medium sodium concentration(0.06 mM from 2.6 mg/L NaF). The second seed culture was incubated for 2days before inoculating 5 mL of the second seed culture into the thirdseed culture containing the same medium and supplements as the secondseed culture. The third seed culture was incubated for 3 days beforeinoculating 5 ml of the third seed culture into the production medium(100 ml in a 500-ml Erlenmeyer flask) with the same composition of theproduction medium, SHY.KcMC, in Example 5.

Per liter of DI H₂O, the production medium consisted of 10 g Starch, 4 gHy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, 100 mg KBr andthe salt supplement shown below. This production medium was thensupplemented with a chemically defined salt formulation and then isreferred to as SHY.KcMC. The chemically defined salt formulationconsisted of:

Salt component Per liter of DI H₂O KCl 30 g CaCl₂•2H₂O 0.43 g SrCl₂ 15.5mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52 μg

About 24 hour after inoculation, sterile XAD-7 resin slurry was added tothe production cultures in a final concentration of 20 g/L. Theproduction culture was then further incubated and sampled at varioustimes to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on a shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby an Agilent HP1100 HPLC using an ACE C-18 reversed-phase column(4.6×150 mm), and an isocratic solvent system consisting of 67% water(0.01% TFA) and 33% acetonitrile (0.01% TFA). The flow rate was 1.5mL/min for 15 min with the detector wavelength set at 210 nm and columntemperature 35° C.

TABLE 13 Production of Salinosporamide A, NPI-0047 and NPI-2065 bySalinispora tropica NPS021184 grown in production medium containing 0.06mM sodium ion based on NaF added to the medium and 11 mM sodium ionbased on ICP-MS analysis. Culture Age Titers (mg/L) (days)Salinosporamide A NPI-0047 NPI-2065 3 45 0 0.7 4 100 0 1.0 5 170 0.5 1.86 176 0.6 2.1

Salinispora tropica NPS21184 can be grown in production mediumcontaining low sodium ion concentration (˜11 mM), which is about 2% ofthe sodium content in sea water (487 mM sodium, see Table 20 of Example9). In this low sodium production medium, Salinispora tropica NPS21184produced 176 mg/L of Salinosporamide A. Since Salinispora tropicaNPS21184 can be grown in the A1-based seed medium with sodium contentestimated at 5.7 mM based on elemental analysis of similar media, thisdemonstrated that Salinispora tropica NPS21184 can be grown in mediumwith sodium content as low as 1% of seawater.

Example 7 Growth of Salinispora tropica NPS021184 in Agar MediaContaining (a) Low Sodium and (b) Low Sodium and Low Chloride

The first seed culture was prepared by inoculating two frozen stocks ofSalinispora tropica NPS021184 into first seed medium consisted ofglucose, Hy Soy, and yeast extract at concentrations of 8 g/L, 6 g/L,and 6 g/L, respectively. The seed medium was then supplemented with 30g/L INSTANT OCEAN® commercial salt formulation.

The first seed culture (100 mL medium containing ˜424 mM sodium ion fromINSTANT OCEAN®) in a 500-mL Erlenmeyer flask was incubated at 28° C. and250 rpm for 72 hours. 1.5 mL of the first seed culture each wastransferred to two sterile 15-mL centrifuge tubes and centrifuged at3,000 rpm for 15 min. The supernatant from each tube was decanted. 10 mLof wash Medium A was added to one tube while 10 mL of Wash Medium B wasadded to the other tube. Both wash media consisted of starch, peptone,and yeast extract at concentrations of 10 g/L, 2 g/L, and 4 g/L,respectively. Wash Medium A also included 30 g/L KCl while Wash Medium Bincluded 48 g/L K₂SO₄. The centrifuge tubes were mixed and thencentrifuged at 3000 rpm for 15 min. The supernatant was decanted and thecells were resuspended in the same Wash Media and centrifuged again aspreviously described. At the final washing, a pipette was used to removemost of the supernatant and to allow enough medium to yield a 2 ml cellsuspension in the tube. These cell suspensions were used as the inoculumfor the agar cultures. Four agar media were used, Agar Media A, A-1, Band B-1, and all consisted of starch, peptone, yeast extract, and agar(Difco, catalog #214530) at concentrations of 10 g/L, 2 g/L, 4 g/L and17 g/L, respectively as well as a salt supplement. Agar Medium A wassupplemented with the following chemically defined salt formulation.This supplemented medium is referred to as A1.Kc4C

Agar Medium A—Salt Supplement Salt component Per liter of DI H₂O KCl 30g MgSO₄•7H₂O 4.29 g KBr 85.9 mg CaCO₃ 0.43 g CaCl₂•2H₂O 429.3 mg SrCl₂15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208 μg

In order to test the significance of ion supplements other thanpotassium, Agar Medium A-1 was supplemented only with 30 g/L KCl. Thatis, to examine if simple replacement of NaCl by KCl in the agar mediumcan support the growth of Salinispora tropica NPS21184.

Agar Medium B was supplemented with the following chemically definedsalt formulation. This supplemented medium is referred to as A1.Ks4C

Agar Medium B—Salt Supplement Salt component Per liter of DI H₂O K₂SO₄48 g MgSO₄•7H₂O 4.29 g KBr 85.9 mg CaCO₃ 0.43 g CaCl₂•2H₂O 429.3 mgSrCl₂ 15.5 g H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208 μg

In order to test the significance of ion supplements other thanpotassium, Agar Medium B-1 was supplemented only with 48 g/L K₂SO₄. Thatis, to examine if simple replacement of NaCl by K₂SO₄ in the agar mediumcan support the growth of Salinispora tropica NPS21184. Particular ionconcentrations of Agar Medium B with the complete salt supplements(Medium A1.Ks4C) were determined by ICP-MS.

TABLE 14A ICP-MS analysis of Particular ion concentrations (mM) inMedium A1.Ks4C, a low sodium and low chloride medium [Na] [Cl] [K] [Mg][Co] [S] [Ca] [Fe] 5.7 14 649 23 1.2 x 10⁻³ 421 4.0 0.011

The sodium and chloride concentration in A1.KsMC are very low at 5.7 mMand 14 mM, respectively, based on ICP-MS analysis. The ion concentrationof A1.KcMC medium was not determined by ICP-MS. However, the sodiumcontent in A1.KcMC should be similar to the sodium content in A1.KsMC,5.7 mM, because the two media have the same composition except A1.KcMChas 30 g/L KCl where A1.KsMC has 48 g/L K₂SO₄.

Sterile inoculation loops were used to transfer cells from Wash Medium Ato agar plates containing 20 mL of Agar Medium A (calculated sodiumcontent: 0.06 mM; calculated chloride content: 415 mM) or 20 mL of AgarMedium A-1 (calculated sodium content: 0.06 mM; calculated chloridecontent: 403 mM). Sterile inoculation loops were used to transfer cellsfrom Wash Medium B to agar plates containing 20 mL of Agar Medium B(calculated sodium content: 0.06 mM; calculated chloride content: 12 mM;ICP-MS analysis: sodium content: 5.7 mM; chloride content: 14 mM) or 20mL of Agar Medium B-1 (calculated sodium content: 0.06 mM; calculatedchloride content: ˜0). After inoculation, the edges of the agar plateswere wrapped with parafilm to avoid evaporation. The agar plates wereincubated at 28° C. for 2 to 4 weeks to observe good growth. No growthwas observed on Agar Medium A-1 and Agar Medium B-1 after 4 weeks ofincubation. Good growth was observed on Agar Medium A and Agar Medium Bafter two weeks of incubation. After two weeks of incubation, the growthfrom Agar Medium A and Agar Medium B were transferred to a fresh platesof Agar Medium A and Agar Medium B, respectively. The re-streaked agarplates were again incubated at 28° C. to observe growth. Good growth onthese re-streaked agar plates were observed in about two to three weeksof incubation and summarized in the table below.

TABLE 14 Observation of growth of Salinispora tropica NPS021184 grown onagar medium A1.Kc4C and Al.Ks4C (second streak) Agar Medium Week 1 Week2 Week 3 Week 4 A1.Kc4C ++ ++ +++ +++ A1.Ks4C −/+ + ++ +++ Key: − nogrowth; −/+ poor growth; + fair growth; ++ growth; +++ very good growth

Example 8 Growth of Salinispora tropica NPS021184 and Production ofNPI-0052 in Media Containing Trace Amounts of Sodium Ion (CalculatedValue Based on 2.6 mg/L NaF: 0.06 mM: ICP-MS Analysis: 9.3 to 11 mM)

Seed cultures were prepared by inoculating a frozen stock of Salinisporatropica NPS021184 into seed medium.

The seed medium consisted of the following per liter of DI water: 10 gStarch, 4 g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, and 40 mg Fe₂(SO₄)₃.The seed medium was then supplemented with a chemically defined saltformulation containing trace amount of sodium (0.06 mM) from NaF. Thissupplemented medium is referred to as SHY.KCD. The composition of thischemically defined salt formulation is shown in the table below

Salt component Per liter of DI H₂O KCl 30 g MgSO₄•7H₂O 4.29 g CaCl₂•2H₂O0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208 μg

The first seed culture (10 mL medium) in a 50-mL culture tube wasincubated for 72 hours before inoculating 5 mL of the first seed cultureinto the second seed medium with the same composition as the first seedmedium including supplemental salts. The second seed culture (100 mLmedium in a 500-mL Erlenmeyer flask) was incubated for 48 hours beforeinoculating 5 mL of the second seed culture into the production medium(100 mL in a 500-mL Erlenmeyer flask) supplemented with trace amount ofsodium ion (0.06 mM from NaF).

Per liter of DI H2O, the production medium consisted of: 10 g Starch, 4g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, 100 mg KBr. Theproduction medium was then supplemented with a chemically defined saltformulation. This supplemented medium is referred to as SHY.KcMC. Thechemically defined salt formulation consisted of:

Salt component Per liter of DI H₂O KCl 30 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52 μg

Because seed medium and production medium were supplemented with 0.06 mMsodium ion derived from NaF (2.6 mg/L). The calculated amount of sodiumion present in the production medium was 0.06 mM. The amount of key ionsin the production medium by ICP-MS analysis is listed in the followingtable.

TABLE 15 Determination of the amount of Particular ions (mM) in MediaSHY.KCD and SHY.KcMC by ICP-MS analysis Medium [Na] [Cl] [K] [Mg] [Co][S] [Ca] [Fe] SHY.KCD 9.3 463 498 20   1 x 10⁻³ 22 3.6 0.12 SHY.KcMC 11477 468 0.75 3.7 x 10⁻⁴ 1.7 4.1 0.12

After about 24 hour incubation into the production culture, sterileXAD-7 resin slurry was added to the production cultures in a finalconcentration of 20 g/L. The production culture was then furtherincubated and harvested at various time points to determineproductivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and an isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min with thedetector wavelength set at 210 nm and column temperature 35° C.

TABLE 16 Production of Salinosporamide A, NPI-0047 and NPI-2065 bySalinispora tropica NPS021184 grown in production medium containing 0.06mM sodium ion (calculated value) and 11 mM sodium ion determined byICP-MS analysis. Titers (mg/L) Culture Salinosporamide Age (days) ANPI-0047 NPI-2065 3 88 0.2 0.4 4 188 0.5 2.4 5 211 0.7 2.8 6 243 0.9 3.7

Salinispora tropica NPS21184 can be grown in medium SHY.KCD containingvery low concentrations of sodium (9.3 mM by ICP-MS analysis) and mediumSHY.KcMC containing very low concentrations of sodium (11 mM by ICP-MSanalysis). Salinispora tropica NPS21184 grown in the above low sodiummedia supported good production of Salinosporamide A (243 mg/L). Thesodium content in SHY.KCD and SHY.KcMC should be very similar with onlyminor differences in the ICP-MS analysis. This minor difference in thesodium content between SHY.KCD and SHY.KcMC is insignificant.

Example 9 ICP-MS Analysis of Trace Elements in Media

Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful toolfor detecting and analyzing trace elements. Over the past few years,ICP-MS has become the technique of choice in many analyticallaboratories for providing the accurate and precise measurements oftrace elements. In ICP-MS, a plasma or gas consisting of ions, electronsand neutral particles, is formed from argon gas, which is then utilizedto atomize and ionize the elements in the sample matrix. These resultingions are the passed through a series of apertures into a high vacuummass analyzer where the isotopes of the elements are identified by theirmass-to-charge ratio. The intensity of a specific peak in the massspectrum is proportional to the amount of the elemental isotope from theoriginal sample.

All samples for trace element analysis were analyzed by inductivelycoupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS) on aPerkin-Elmer ELAN DRC II. Aliquots of each sample are introduced into aradio frequency (RF) plasma where energy-transfer processes causedesolvation, atomization, and ionization. The ions are extracted fromthe plasma through a differentially-pumped vacuum interface and travelthrough a pressurized chamber (DRC) containing a specific reactive gaswhich preferentially reacts with interfering ions of the same targetmass to charge ratios (m/z). A solid-state detector detects ionstransmitted through the mass analyzer, on the basis of theirmass-to-charge ratio (m/z), and the resulting current is processed by adata handling system.

Different reaction gases and settings are applied depending on thetarget analyte and projected interference. Comparison of the differentisotopes, reaction gases, and reaction gas settings allow forinterference monitoring and selection of optimum instrument settingsdepending on each sample matrix type and element.

Instrument Parameters

Tables 17, 18, and 19 show the ICP-DRC-MS operating conditions for thedifferent elements.

TABLE 17 ICP-DRC-MS Operating Conditions and Parameters ParameterSetting/Type Nebulizer Meinhard Type A Quartz RF Power 1200W Plasma ArFlow 15 L/min Nebulizer Ar Flow 0.87 L/min Injector 2.0 mm I.D. QuartzMonitored ²³NaN⁺, ³⁹KN⁺, ²⁴Mg⁺, ²⁶Mg⁺, ⁴⁰Ca⁺, ⁴³Ca⁺, ion (m/z) ⁴⁴Ca⁺,⁵⁴Fe⁺, ⁵⁶Fe⁺, Reaction Gas NH₃ NH3 Flow 0.8 mL/min RPq 0.7

TABLE 18 ICP-DRC-MS Operating Conditions and Parameters ParameterSetting/Type Nebulizer Meinhard Type A Quartz RF Power 1200W Plasma ArFlow 15 L/min Nebulizer Ar Flow 0.87 L/min Injector 2.0 mm I.D. QuartzMonitored ion m/z) ⁴⁸SO⁺, ⁵⁰SO⁺ Reaction Gas NH₃ O2 Flow 0.9 mL/min RPq0.7, 0.75, 0.6

TABLE 19 ICP-DRC-MS Operating Conditions and Parameters ParameterSetting/Type Nebulizer Meinhard Type A Quartz RF Power 1200W Plasma ArFlow 15 L/min Nebulizer Ar Flow 0.87 L/min Injector 2.0 mm I.D. QuartzMonitored ion (m/z) ³⁵Cl⁺, ³⁷Cl⁺ Reaction Gas none RPq 0.25

The complex medium components, such as yeast extract and Hy Soy, areknown to contain trace elements such as sodium, potassium, magnesium andchloride. Therefore, adding these nutrients into the medium introducesthe above trace elements to the growth medium. This phenomenon is knownas carryover effect. Therefore, even without addition of the salt of theabove elements to the growth medium, the growth medium can contain theabove elements contributed from the complex nutrient such as yeastextract and Hy Soy. In order to determine the carryover effect by yeastextract, Hy Soy and the other nutrient components, we use ICP-MStechnique to analyze the presence of the trace elements present in thefollowing media:

TABLE 20 Determination of the amount of Particular ions (mM) in Mediaand seawater by ICP-MS analysis Media [Na] [Cl] [K] [Mg] [Co] [S] [Ca][Fe] 4 g/L Yeast Extract (USB 23547, 1.8 ND 3.4 0.10 6 x 10⁻⁵ 2.5 NT NTlot # 117942) in deionized water 4 g/L Hy Soy (Kerry Biosciences 4.9 ND2.8 0.34 1 x l0⁻⁵ 2.1 NT NT 5X59089, lot # M031936) in deionized water 4g/L. Yeast Extract (USB 23547, 8.3 3.6 8.9 0.56 7 x 10⁻⁵ NT NT NT lot #117942) + 4 g/L Hy Soy (Kerry Biosciences 5X59089, lot # M031936) indeionized water 10 g/L Starch (USB 21695, lot # 7.7 4.6 7.3 0.63 6 x10⁻⁵ 1.5 0.42 0.012 118386) + 4 g/L Yeast Extract (USE 23547, lot #117942) + 4 g/L Hy Soy (Kerry Biosciences 5X59089, lot # M031936) indeionized water Medium SHY.10.4.4* in 7.6 4.7 7.2 0.61 7 x 10⁻⁵ 2.7 0.840.17 deionized water Medium SHY.10.4.4* + 24 g/L 416 505 7.0 0.69 7 x10⁻⁵ 3.4 1.2 0.17 NaCl in deionized water Medium SHY.10.4.4* + In house426 507 15 0.64 0.45 3.3 1.2 0.17 salt formulation I (production)**Instant Ocean (30 g/L) 424 542 8.6 47 ND 31 NT NT A1*** in deionizedwater 5.2 ND 4.2 0.19 6 x 10⁻⁵ 0.39 0.08 0.008 Seawater**** 487 530 1061 5 x 10⁻⁵ 32 11 2 x 10⁻⁴ NT = Not Tested ND = Not Detected (belowdetection limit) *Medium SHY.10.4.4 10 g/L Starch (USB 2.1695) 4 g/L HySoy (Kerry Biosciences M031936) 4 g/L Yeast Extract (USB 23547) 40 mg/LFerric sulfate 100 mg/L KBr 1 g/L Calcium carbonate **In-house saltformulation I (Production) 24 g/L NaCl 686.8 mg/L KCl 429.3 mg/LCaCl₂·2H₂O 15.5 mg/L SrCl₂ 21.5 mg/L H₃BO₃ 2.6 mg/L NaF ***Medium A1 10g/L Starch (USB 21695) 2 g/L Peptone (USB 20048) 4 g/L Yeast extract(USB 23547) ****Seawater from the Pier of University of California atSan Diego, La Jolla, California.

It is clear from the above ICP-MS analysis that the medium componentsfrom SHY. 10-4-4 in deionized water, and a combination of starch, Hy Soyand yeast extract, the medium components contribute 7.6 to 8.3 mM ofsodium to the growth medium without addition of any sodium salt to thegrowth medium. The major source of carryover of sodium is Hy Soy. A 4g/L solution of this media component contained 4.9 mM of sodium. Thecarryover of chloride from the medium components is about 3.6 to 4.7 mM.For the A1 medium in deionized water, the carryover of sodium is about5.2 mM. The sodium content of the seawater collected at the Pier of theUniversity of California at San Diego, La Jolla, Calif. is 487 mM byICP-MS analysis.

Example 10 Preparation of Low Sodium Frozen Vegetative Stock Salinisporatropica CNB440, CNB476 and NPS021184 in Seed Medium Containing TraceAmount of Sodium Ion (0.06 mM Calculated Value; ˜9-10 mM Determined byICP-MS Analysis)

To prepare the Low Sodium Frozen Stock, the first seed culture wasprepared by inoculating two frozen stock vials of each strain ofSalinispora tropica CNB440, CNB476 and NPS021184 (previously prepared in2.4% NaCl medium or 3% INSTANT OCEAN® commercial salt formulationmedium) into seed medium consisting of the following per liter of DIH₂O: 10 g Starch, 4 g Hy Soy, 4 g Yeast Extract, 40 mg Fe₂(SO₄)₃. Thisseed medium was then supplemented with a chemically defined saltformulation. The chemically defined salt formulation included. The seedmedium with this salt supplement is the SHY.B4C medium.

Salt component Per liter of DI H₂O NaCl 24 g KCl 0.69 g MgSO₄•7H₂O 4.29g CaCl₂•2H₂O 0.43 g SrCl₂ 155 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208μg

The first seed culture (100 mL medium) in a 500-mL Erlenmeyer flask wasincubated for 72 hours at 250 rpm and 28° C. 45 mL each of the firstseed cultures was transferred to a sterile 50-mL centrifuge tube. Thecentrifuge tubes were centrifuged at 3,500 rpm for 10 min. Thesupernatant was discarded and 40 mL of the wash medium was added to eachcentrifuge tube. The Wash Medium consisted of the following per liter ofDI H₂O: 10 g Starch, 4 g Hy Soy, 4 g Yeast Extract, 40 mg Fe₂(SO₄)₃, 30g KCl. The centrifuge tubes were gently reverted several times to reusedthe pellet into the Wash Medium and the centrifuge tubes werecentrifuged at 3,500 rpm for 10 min. The supernatant was discarded andthe centrifuge wash protocol was repeated with fresh Wash Medium. Afterdiscarding the supernatant at the end of the second wash, 45 mL of freshWash Medium was added to each centrifuge tube to re-suspend the culturebefore transferring together to a sterile 500 mL Erlenmeyer flask. FivemL of Wash Medium was used to rinse each centrifuge tube and the rinseswere added to the cell suspension in the 500-mL Erlenmeyer flask (totalvolume of the cell suspension was ˜100 mL). The washing procedure servedto remove the sodium chloride, more specifically, the sodium ion, thatwas present in the first seed medium. Five mL of each of the washedcultures was inoculated into a second stage seed flask containing mediumwith the following composition per liter of D.I. water, two flasks wereprepared for each strain: 10 g Starch, 4 g Hy Soy, 4 g Yeast Extract, 1g CaCO₃, and 40 mg Fe₂(SO₄)₃. The two second seed media were thensupplemented with a chemically defined salt formulation containing verylow concentration of 0.06 mM sodium ion derived from NaF. The chemicallydefined salt formulation included (per liter of DI H₂O):

SHY.KCA SHY.KCD 30 g KCl 30 g KCl 429 g MgSO₄•7H₂O 4.29 g MgSO₄•7H₂O0.43 g CaCl₂•2H₂O 15.5 mg SrCl₂ 21.5 mg H₃BO₃ 2.6 mg NaF 208 μgCoCl₂•6H₂O

The second seed cultures were incubated for 24 to 96 hours at 250 rpmand 28° C. to achieve the following dry cell weights:

TABLE 21 Dry cell weight of Salinispora tropica CNB440, CNB476 andNPS21184 grown in Media SHY.KCA and SHY.KCD Dry Cell Weight (mg/mL)Strain Medium 24h 48h 72h 96h CNB440 SHY.KCA 1.39 1.36 1.57 1.56 SHY.KCD2.55 1.99 2.55 2.68 CNB476 SHY.KCA 1.43 1.15 1.18 1.52 SHY.KCD 2.17 2.192.07 2.35 NPS21184 SHY.KCA 0.73 3.51 5.50 3.41 SHY.KCD 2.60 3.27 5.394.13

Strains CNB440 and CNB476 grew better in the SHY.KCD medium than in theSHY.KCA medium. SHY.KCD medium has more ion supplements than SHY.KCA.These ion supplements are responsible for supporting better grow ofstrains CNB440 and CNB476. The initial growth rate of strain NPS21184was faster in the SHY.KCD medium than in the SHY.KCA medium. The growthyield of strain NPS21184 is similar in SHY.KCA and SHY.KCD media. StrainNPS21184 has better growth rate and yield than strains CNB440 andCNB476. After 48 hours of incubation, 5 mL each of the second seedcultures of NPS21184 were inoculated into the corresponding third seedmedia SHY.KCA and SHY.KCD, with the same composition as the second seedmedia. Since the growth of strains CNB440 and CNB476 in medium SHY.KCAwas poor, the cultures growing in medium SHY.KCA were not moved forwardfor preparation of freeze stock. In order to compensate the lower ingrowth yield of strains CNB440 and CNB476 in SHY.KCD medium, 45 mL eachof the 96-hour seed cultures were centrifuged in a sterile 50-mLcentrifuge tube. 35 mL of the supernatant was withdrawn from thecentrifuge tube to concentrate the culture by 4.5 fold (from 45 mL to 10mL culture). 5 mL each of the concentrated cultures of CNB440 and CNB476in KCD was used to inoculate into the third seed medium SHY.KCD. Thethird seed cultures (100 mL medium in a 500-mL Erlenmeyer flask) wereincubated for 24 hours to 120 hours at 250 rpm and 28° C. to achieve thefollowing dry cell weight.

TABLE 22 Dry cell weight of Salinispora tropica CNB440, CNB476 andNPS21184 grown in Media SHY.KCA and SHY.KCD Dry Cell Weight (mg/mL)Strain Medium 24 h 48 h 72 h 96 h 120 h CNB440 SHY.KCD 1.63 2.10 2.582.79 2.73 CNB476 SHY.KCD 1.78 1.68 1.23 1.98 2.77 NPS21184 SHY.KCA 2.103.87 0.0 0.07 ND* SHY.KCD 1.91 6.16 6.57 7.77 6.03 *ND = Not Determined

Strain NPS21184 grew very well in SHY.KCD medium with the majority ofthe growth completed at 48 hours (6.16 mg/L dry cell weight) andyielding the highest growth yield of 7.77 mg/L dry cell weight. Sincegood growth rate of NPS21184 in SHY.KCD medium was observed, 8 mL eachof the 48-hour third seed culture was added to a sterile 50-mL culturetube (25×150 mm) containing 2 mL of 50% glycerol. The culture tubes wereincubated for one hour at 250 rpm and 28° C. 1.5-mL portions of theculture were transferred to sterile cryovials (1.8 mL capacity). Thefreeze stock is stored at −80° C. freezer until use. Alternatively, theculture tube containing the culture in 10% glycerol is stored at −80° C.freezer until use. We have successfully prepared the frozen freeze stockof strain NPS21184 in SHY.KCD medium that contains very low sodiumcontent (9.3 mM sodium by ICP-MS analysis, see table below).

Strain NPS21184 grew well in SHY.KCA medium, which has the same sodiumconcentration as the SHY.KCD medium (see table below) for the first 48hours incubation yielding a dry cell weight of 3.87 mg/L. However, theculture lost viability with further incubation in SHY.KCA medium,indicating again that the importance of the additional ion supplements,besides potassium and magnesium, in SHY.KCD medium for supporting growthof Salinispora tropica in low sodium medium. No frozen stock of strainNPS21184 in SHY.KCA medium was prepared.

To prepare freeze stocks of strains CNB440 and CNB476 in KCD medium, the120-hour cultures were first concentrated 2.3-3.9 fold to compensate forthe low cell yield. Four×35 mL cultures each of strains CNB440 andCNB476 in 50-mL centrifuge tubes were centrifuged at 3,000 rpm for 15min. Twenty mL supernatant from each tube of strain CNB440 was withdrawnand the concentrated cultures were combined to yield ˜45 mL ofconcentrated cultures (˜2.3 fold concentrate). Twenty-six mL supernatantfrom each tube of strain CNB476 was withdrawn and the concentratedcultures were combined to yield ˜36 mL of concentrated cultures (˜3.9fold concentrate). Eight mL each of the concentrated 120-hour third seedculture was added to a sterile 50-mL culture tube (25×150 mm) containing2 mL of 50% glycerol. The culture tubes were incubated for one hour at250 rpm and 28° C. 1.5-mL portions of the culture were transferred tosterile cryovials (1.8 mL capacity). The freeze stock was stored at −80°C. freezer until use. Alternatively, the culture tube containing theculture in 10% glycerol is stored at −80° C. freezer until use. LowSodium Frozen Stocks of strains NPS429 and NPS465 in SHY.KCD medium havebeen prepared which contain very low sodium content (9 to 10 mM sodiumby ICP-MS analysis, see Table below).

TABLE 23 Determination of the amount of Particular ions (mM) present inthe media by ICP-MS analysis Media [Na] [Cl] [K] [Mg] [Co] [S] [Ca] [Fe]l^(st) Seed 413 493 6.1 21 1 × 10⁻³ 23 3.9 0.17 Medium (SHY.B4C) Wash7.1 494 434 0.62 7 × 10⁻⁵ 3.8 1.4 0.17 Medium SHY.KCA 10 487 499 22 7 ×10⁻⁵ 22 1.1 0.14 SHY.KCD 9.3 463 498 20 1 × 10⁻³ 22 3.6 0.12

Even though the only sodium salt added to the Wash Medium SHY.KCA andSHY.CKD is sodium fluoride, which supplies 0.06 mM sodium to the abovemedia, ICP-MS analysis of these media showed that the sodiumconcentration in these media were 9 to 10 mM. The majority of the sodium(˜8 mM) in these media was derived from yeast extract and Hy Soy (seeTable 20 in Example 9).

Media containing low sodium (9 to 10 mM, ˜2% of the sodium content ofseawater) were developed to support the growth of Salinispora tropicaCNB440, CNB476 and NPS21184 for the preparation of low sodium vegetativefrozen stock of these Salinispora tropica strains.

Example 11 Growth of Salinispora tropica NPS021184 and Production ofSalinosporamide A in Seed and Production Media Containing Trace Amountof Sodium Ion (0.06 mM, Calculated Value; 9 to 10 mM by ICP-MS Analysis)Using Low Sodium Frozen Stock

In order to confirm the growth and the productivity of the Low SodiumFrozen Stock of NPS21184 prepared in the low sodium medium SHY.KCD, thefrozen stock prepared in Example 10 was used to inoculate 10 mL each ofthe following seed media (per liter of DI H₂O) in a 50-mL culture tube(25×150 mm):

SHY.B4C SHY.KCA SHY.KCD 10 g Starch 10 g Starch 10 g Starch 4 g Hy Soy 4g Hy Soy 4 g Hy Soy 4 g Yeast Extract 4 g Yeast Extract 4 g YeastExtract 40 mg Fe₂(SO₄)₃ 40 mg Fe₂(SO₄)₃ 40 mg Fe₂(SO₄)₃ 24 g NaCl 30 gKCl 30 g KCl 0.69 g KCl 4.29 g MgSO₄•7H₂O 4.29 g MgSO₄•7H₂O 4.29 gMgSO₄•7H₂O 1 g CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g CaCl₂•2H₂O — 15.5 mg SrCl₂15.5 mg SrCl₂ — 21.5 mg H₃BO₃ 21.5 mg H₃BO₃ — 2.6 mg NaF 2.6 mg NaF —208 μg CoCl₂•6H₂O 208 μg CoCl₂•6H₂O — 1 g CaCO₃

While the amount of known sodium salt, sodium fluoride, added to themedia SHY.KCA and SHY.KCD was 2.6 mg/L (0.06 mM sodium), ICP-MS analysisdemonstrated that the sodium concentration in KCA and KCD were 9 and 10mM, respectively (see table 23 of Example 10). The sodium content inMedium SHY.B4C was 413 mM, by ICP-MS analysis. Media SHY.KCA and SHY.KCDcontained very low concentration of sodium, about 40-fold less thanMedium SHY.B4C.

1.8 mL of the frozen stock each was used to inoculate 10.2 mL first seedmedium SHY.B4C and medium SHY.KCA (total volume: 12 mL) to yield aninoculum size of 15%. 1.2 mL, 2.4 mL and 3.6 mL of the frozen stock wereinoculated into 10.8 mL, 9.6 mL and 8.4 mL of first seed medium SHY.KCD(total volume: 12 mL) to yield inoculums size of 10%, 20% and 30%,respectively. 1.8-mL of frozen stock of NPS21184 (i.e., prepared in 24g/L NaCl medium or 30 g/L INSTANT OCEAN® commercial salt medium) wasalso used to inoculate 10.2 mL first seed medium SHY.B4C as a controlcondition of high sodium medium (saline fermentation condition). Thefirst seed cultures were incubated for 72 hours at 250 rpm and 28° C.yielding the following dry cell weight data.

TABLE 24 Dry cell weight of first seed cultures (72 hours) [Sodium] inFreeze % Dry Cell Weight Preparation* Inoculum [Sodium] in Medium.*(mg/mL) A) 426 mM**  15% SHY.B4C (413 mM) 4.2 B) 9.3 mM  15% SHY.B4C(413 mM) 4.4 C) 9.3 mM 1.5% SHY.KCA (10 mM) 2 D) 9.3 mM  10% SHY.KCD(9.3 mM) 4.4 E) 9.3 mM  20% SHY.KCD (9.3 mM) 4.6 F) 9.3 mM  30% SHY.KCD(9.3 mM) 4.6 *Sodium concentration of SHY.B4C, SHY.KCA and SHY.KCD wasdetermined by ICP-MS analysis (Table 23 of Example 10) **Sodiumconcentration was estimated from the condition of Medium SHY.10.4.4 + 24g/L NaCl in deionized water in Table 20 of Example 9

It is clear from the dry cell weight data that the freeze preparation ofNPS21184 in the low sodium medium can support the good growth yield inMedium SHY.KCD, a low sodium medium that is supplemented with tracemetal ions other than sodium. Without the addition of other ions inMedium SHY.KCA with similar sodium concentration as medium SHY.KCD, thegrowth yield was significantly reduced by more than 50%. It is alsoimportant to observe that the Low Sodium Frozen Stock prepared in thelow sodium medium SHY.KCD (9.3 mM) has the same growth yield as thefrozen stock prepared in the high sodium medium (426 mM in NaCl basedmedium).

After incubating the above first seed cultures for 72 hours at 250 rpmand 28° C., 0.5 mL of each culture was inoculated into 10 mL of thesecond seed medium (in 50-mL culture tubes) having the same compositionas the first seed medium. The second seed cultures were incubated for 72to 96 hours and yielding the following daily dry cell weight data:

TABLE 25 Dry cell weight data for second seed cultures [Sodium] inFreeze % Inoculum Dry cell weight (mg/mL) Preparation* Medium 1^(st)seed 2^(nd) seed 24 h 48 h 72 h 96 h A) 426 mM** SHY.B4C 15% 5% 2.6 3.64.4 4.2 B) 9.3 mM SHY.B4C 15% 5% 1.8 2.8 3.9 4.8 C) 9.3 mM SHY.KCA 15%5% 0.49 1.0 0.65 ND D) 9.3 mM SHY.KCD 10% 5% 1.4 3.2 3.4 ND E) 9.3 mMSHY.KCD 20% 5% 1.9 2.9 3.9 4.2 F) 9.3 inM SHY.KCD 30% 5% 1.1 3.6 3.2 4.4*Sodium. content determined by ICP-MS analysis **Sodium concentrationestimated from the condition of Medium SHY.10.4.4 + 24 g/L NaCl indeionized water in Table 20 of Example 9 ND = Not determined

The dry cell weight data from the second seed cultures again support thefinding that the Low Sodium Frozen Stock preparation of NPS21184 cansupport good cell growth in low sodium medium (Medium SHY.KCD) whichincludes trace metal ions other than sodium. The Low Sodium Frozen Stockhas the same growth yield as the frozen stock prepared in a high sodiummedium. The importance of the metal ions present in Medium SHY.KCD iseven more evident in that the second seed culture with Medium SHY.KCAyielded lower dry cell weight than the first seed culture. The dry cellweight of SHY.KCA medium in the second seed culture was only about 25%of the SHY.KCD medium.

To determine the production profile of Low Sodium Frozen Stock ofNPS21184 grown up in low sodium medium (Medium SHY.KCD), 0.5 mL of the48-hour sample of the second seed culture from Condition D in Table 25(l seed inoculum: 10%; second seed inoculum: 5%; both in Medium SHY.KCD)was used to inoculate 10 mL of Medium SHY.KCD in 50-mL culture tubes.Five mL of the 48-hour sample of the second seed culture of controlCondition A in Table 25 (1^(st) seed inoculum: 15%; 2^(nd) seedinoculum: 5%; both in Medium SHY.B4C) was also used to inoculate 10 mLof Medium SHY.B4C in 50-mL culture tubes. The production cultures wereincubated at 250 rpm and 28° C.

About 24 hour after inoculation, sterile XAD-7 resin slurry was added tothe production cultures in a final concentration of ˜30 g/L. Theproduction culture was then further incubated and harvested at varioustime points to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. before HPLC analysis.The dried extract was resuspended in 320 μL DMSO and injected into HPLCusing the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

TABLE 26 Comparison of production of Salinosporamide A, NPI-0047 andNPI-2065 by Salinispora tropica NPS021184 grown in production mediumcontaining high and low sodium concentration. [Sodium] in seed [sodium]and Day 4 Titers (mg/L) Day 5 Titers (mg/L) Day 6 Titers (mg/L) infreeze production NPI- NPI- NPI- NPI- NPI- NPI- prep* media* 0047 2065Sal A 0047 2065 Sal A 0047 2065 Sal A 426 mM  413 mM 0.52 6.7 261 0.978.4 324 0.73 8.2 310 9.3 mM  9.3 mM 0.6 2.4 163 0.77 3.9 242 0.50 3.6209 *Sodium content determined by ICP-MS analysis

The maximum production of Salinosporamide A by NPS21184 in the lowsodium medium (9.3 mM sodium, SHY.KCD) was about 75% of the high sodiummedium (413 mM sodium, SHY.B4C). The above data demonstrates that strainNPS21184 can be grown in low sodium medium (9.3 mM) with good cellgrowth and produce substantial quantities of Salinosporamide A. Thisdata showed about 75% of the highest yield of the high sodium (413 mM),saline fermentation condition.

Example 12 Comparison of Growth of Low Sodium Frozen Stock ofSalinispora tropica CNB440, CNB476 and NPS021184 in Low Sodium Mediumand Production Profiles of these Strains in Low Sodium Medium

Low Sodium Frozen Stocks of Salinispora tropica CNB440, CNB476 andNPS021184 prepared in example 10 were used to inoculate 10 mL each ofthe following seed medium SHY.KCD in a 50-mL culture tube (25×150 mm).

SHY.KCD (per liter of DI H₂O)

10 g Starch 4 g Hy Soy

4 g Yeast extract

1 g CaCO₃ 40 mg Fe₂(SO₄)₃ 0.69 g KCl 4.29 g MgSO₄.7H₂O 0.43 g CaCl₂.2H₂O15.5 mg SrCl₂ 21.5 mg H₃BO₃ 2.6 mg NaF 208 μg CoCl₂.6H₂O

1 mL of the frozen stock each was used to inoculate 10 mL first seedSHY.KCD medium to yield an inoculum size of 10%. After incubating theabove first seed cultures of CNB476 and NPS21184 for 72 hours at 250 rpmand 28° C., 5 mL of each culture was inoculated into 100 mL of thesecond seed medium (in 500-mL Erlenmeyer flask) having the samecomposition as the first seed medium. For CNB440, the first seed culturewas grown for 5 days. 10 mL of the first seed culture was used toinoculate 100 mL of the second seed medium in a 500-mL Erlenmeyer flask.The second seed cultures were incubated for 48 to 96 hours and yieldingthe following dry cell weight data:

TABLE 27 Dry cell weight of Salinispora tropica CNB440, CNB476 andNPS21184 in low sodium (9.3 mM) medium SHY.KCD Dry cell weight (mg/m)LStrain 48 hours 72 hours 96 hours NPS21184 5.7 4.9 5.3 CNB476 5.3 6.37.5 CNB440 2.3 4.0 5.2

The dry cell weight data from the second seed cultures confirmed thefinding that the Low Sodium Frozen Stocks of CNB440, CNB476 and NPS21184prepared in the low sodium medium (9.3 mM) can support good cell growthin low sodium seed medium that is supplemented with trace metal ionsother than sodium. The Low Sodium Frozen Stocks of strains CNB440,CNB476 and NPS21184 grew very well in the low sodium medium (9.3 mMsodium) with dry cell weights of 5.2 to 7.5 mg/mL. While the growth rateof CNB440 is slower in the low sodium medium, it achieved the same drycell weight as NPS21184 after 96 hours incubation.

To determine the production profile of Low Sodium Frozen Stocks culturesof CNB440, CNB476 and NPS21184 grown up in low sodium production medium,5 mL each of the 48-hour second seed culture samples of CNB476 andNPS21184 was used to inoculate 100 mL of production medium having thesame composition as the seed medium in a 500-mL Erlenmeyer flasks. ForCNB440, 5 mL of the 72 hour second seed culture sample was used toinoculate 100 mL of production medium having the same composition as theseed medium in a 500-mL Erlenmeyer flasks. The production cultures wereincubated at 250 rpm and 28° C.

About 24 hour after inoculation, sterile XAD-7 resin slurry was added tothe production cultures in a final concentration of ˜20 g/L. Theproduction culture was then further incubated and harvested at varioustime points to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

TABLE 28 Production of NPI-0052, NPI-0047 and NPI-2065 by Salinisporatropica NPS021184 grown in production medium containing low sodiumconcentration (9.3 mM by ICP-MS analysis), Titers (mg/L) Day 3 Day 4 Day5 Day 6 NPI- NPI- NPI- NPI- NPI- NPI- NPI- NPI- Strain Sal A 0047 2065Sal A 0047 2065 Sal A 0047 2065 Sal A 0047 2065 21184 97 0.14 0.79 1910.86 3.0 180 0.48 2.8 201 0.62 3.4

TABLE 29 Production of Salinosporamide A, NPI-0047 and NPI-2065 bySalinispora tropica NPS465 grown in production medium containing lowsodium concentration (9.3 mM by ICP-MS analysis). Titers (mg/L) Day 4Day 5 Day 6 Day 7 NPI- NPI- NPI- NPI- NPI- NPI- NPI- NPI- Strain Sal A0047 2065 Sal A 0047 2065 Sal A 0047 2065 Sal A 0047 2065 465 118 3.70.73 128 3.5 0.90 130 3.8 0.89 124 3.3 0.77

TABLE 30 Production of Salinosporamide A, NPI-0047 and NPI-2065 bySalinispora tropica NPS429 grown in production medium containing lowsodium concentration (9.3 mM by ICP-MS analysis). Titers (mg/L) Day 4Day 5 Day 6 Day 7 NPI- NPI- NPI- NPI- NPI- NPI- NPI- NPI- Strain Sal A0047 2065 Sal A 0047 2065 Sal A 0047 2065 Sal A 0047 2065 429 109 1.80.35 126 2.7 0.74 158 2.9 1.3 156 2.9 1.3

We detected production of Salinosporamide A, NPI-0047 and NPI-2065 inall three Salinispora tropica strains grown in low sodium medium withNPS21184 showing the highest concentration of Salinosporamide A at 201mg/L. The production of NPI-0047 was significantly lower in strainNPS21184 than the other two strains.

Example 13 Growth of Low Sodium Frozen Stocks of Salinispora tropicaCNB440, CNB476 and NPS21184 on Agar Medium Containing Low Concentrationof Sodium (9.3 mM by ICP-MS Analysis)

Low Sodium Frozen Stocks of Salinispora tropica CNB440, CNB476 andNPS21184 prepared in Example 10 in low sodium medium containing 9.3 mMsodium (by ICP-MS analysis) were tested for growth on agar media alsocontaining low sodium concentration (SHY.KCX, SHY.KCY and TCG.KCX). Agarplates containing high concentrations of sodium (saline fermentationcondition, SHY.IO and TCG.B4C) and agar plates with low sodium (carbonand nitrogen sources provided in DI water; but with no salt supplementor FeSO₄ only—SHY.DI and TCG.DI media) were also included in this studyfor comparison purpose. After thawing the Low Sodium Frozen Stocks atroom temperature, 30 L each of the cultures were spread onto agar plates(60×15 mm, 10 mL agar medium) with the compositions listed in the tablebelow.

(Per liter of Di water) SHY.IO SHY.KCX SHY.KCY SHY.D1 TCG.B4C TCG.KCXTCG.DI 10 g Starch 10 g Starch 10 g Starch 10 g Starch 10 g Starch  4 gGlucose  4 g Glucose  4 g Hy Soy  4 g Hy Soy  4 g Hy Soy  4 g Hy Soy  4g Hy Soy  3 g Tryptone  3 g Tryptone  4 g Yeast  4 g Yeast  4 g Yeast  4g Yeast  4 g Yeast  5 g Casitone  5 g Extract Extract Extract ExtractExtract Casitone 40 mg 40 mg 40 mg 40 mg 40 mg 30 g KCI 17 g AgarFe₂(SO₄)₃ Fe₂(SO₄)₃ Fe₂(SO₄)₃ Fe₂(SO₄)₃ Fe₂(SO₄) 30 g Instant 30 g KCl30 g KCl 17 g Agar 24 g NaCl 4.29 g Ocean MgSO₄•7H₂O 17 g Agar 4.29 g0.43 g 4.29 g 0.43 g MgSO₄•7H₂O CaCl₂•2H₂O MgSO₄•7H₂O CaCl₂•2H₂O 0.43 g15.5 mg SrCl₂ 0.43 g 15.5 mg CaC1₂•2H₂O CaCl₂•2H₂O SrCl₂ 15.5 mg 21.5 mg15.5 mg 21.5 mg SrCl₂ H₃BO₃ SrCl₂ H₃BO₃ 21.5 mg  2.6 mg NaF 21.5 mg  2.6mg NaF H₃BO₃ H₃BO₃ 2.6 mg NaF 208 μg  2.6 mg NaF 208 μg CoCl₂•6H₂OCoC1₂•6H₂O 208 μg 17 g Agar 208 μg 17 g Agar CoCl₂•6H₂O CoCl₂•6H₂O 17 gAgar 17 g Agar

The sodium content of the above agar media was determined by ICP-MSanalysis:

TABLE 31 Sodium content determined by ICP-MS analysis (without the agar)SHY.IO SHY.KCX SHY.DI TCG.B4C TCG.KCX TCG.DI [Na] 452 mM 10 mM 7.6 mM ND11 mM 11 mM No ICP-MS analysis of SHY.KCY sample but the sodium contentshould be the same as SHY.KCX. ND = Not Determined

After inoculation, the edges of the agar plates were wrapped withparafilm to avoid evaporation and the plates were incubated at 28° C.for 4 weeks. The growth of Salinispora tropica CNB440, CNB476 andNPS21184 on the agar plates are summarized below:

TABLE 32 Salinisvora tropica CNB440, 1^(st) streak SHY.IO SHY.KCXSHY.KCY SHY.DI TCG.B4C TCG.KCX TCG.DI Wk 1 + − − − + −/+ − Wk 2 ++ −/+ −− ++ ++ − Wk 3 +++ −/+ − − +++ +++ − Wk 4 +++ + −/+ − +++ +++ − Key: −no growth; −/+ poor growth; + fair growth; ++ good growth; +++ goodgrowth

TABLE 33 Salinispora tropica CNB476, 1^(st) streak SHY.IO SHY.KCXSHY.KCY SHY.DI TCG.B4C TCG.KCX TCG.DI Wk 1 ++ −/+ − − ++ + − Wk 2 +++−/+ −/+ − +++ +++ − Wk 3 +++ −/+ −/+ − +++ +++ − Wk 4 +++ + −/+ − ++++++ − Key: − no growth; −/+ poor growth; + fair growth; ++ good growth;+++ good growth

TABLE 34 Salinispora tropica NPS21184, 1^(st) streak SHY.IO SHY.KCXSHY.KCY SHY.DI TCG.B4C TCG.KCX TCG.DI Wk 1 ++ + −/+ − ++ + − Wk 2 +++ +−/+ − +++ ++ − Wk 3 +++ + −/+ − +++ +++ − Wk 4 +++ + −/+ − +++ +++ −Key: − no growth; −/+ poor growth; + fair growth; ++ good growth; +++good growth

Good growth was observed for all three strains growing on agar mediacontaining high sodium (saline fermentation conditions with sodiumconcentration of 452 mM::SHY.IO and TCG.B4C). No growth was observed forany strain on low sodium media (SHY.DI and TCG.DI). The sodium contentin these media is very low, at 7.6 mM (SHY.DI) to 11 mM (TCG.DI) byICP-MS analysis. We observed growth of all three Salinispora tropicastrains on agar media containing low level of sodium but supplementedwith 30 g/L KCl and other ions (SHY.KCX and TCG.KCX). The SHY.KCX andTCG.KCX media contain similar sodium content as the SHY.DI and TCG.DI,about 9 to 11 mM. This demonstrates the importance of adding KCl andother ions to the medium for supporting the growth of Salinisporatropica. The growth of all three Salinispora tropica strains on theTCG.KCX agar is significantly better than the SHY.KCX agar even thoughthe sodium and other ion contents in these agar media are very similar.This also demonstrates the importance of the proper composition of agarmedium in supporting the growth of Salinispora tropica strains. Thisobservation is also substantiated by the observation of good growth ofSalinispora tropica NPS021184 on A1.Kc4C agar medium in Example 7. A1based agar medium is as good as TCG-based agar medium in supporting thegrowth of Salinispora tropica NPS21184 under low sodium condition. Wealso observed that the growth in SHY.KCX medium is better than thegrowth in the SHY.KCY medium without addition of magnesium. Thisindicated the importance of magnesium in supporting the growth ofSalinispora tropica. We demonstrate here that Salinispora tropica can begrown in agar media containing low levels of sodium (10 to 11 mM) andsupplemented with KCl and other ions (non-saline condition) shown in thetable above.

To confirm the growth of Salinispora tropica strains in media containinglow level of sodium (non-saline condition), two-week old agar culturesgrowing on SHY.KCX and TCG.KCX media were re-streaked onto thecorresponding agar media by the sterile inoculation loops. The two-weekold agar cultures growing on high sodium condition (SHY.IO and TCG.B4C)were also re-streaked onto the corresponding agar media for comparisonpurpose. After inoculation, the edges of the agar plates were wrappedwith parafilm to avoid evaporation and the plates were incubated at 28°C. for 4 weeks. The growth of Salinispora tropica CNB440, CNB476 andNPS21184 on the agar cultures are summarized below:

TABLE 35 Salinipora tropica CNB440, 2^(nd) streak SHY.IO SHY.KCX TCG.KCXTCG.B4C Week 1 +++ −/+ ++ ++ Week 2 +++ ++ +++ ++ Week 3 +++ ++ +++ +++Week 4 +++ ++ +++ +++ Key: − no growth; −/+ poor growth; + fair growth;++ good gowth; +++ very good growth

TABLE 36 Salinipora tropica CNB476, 2^(nd) streak SHY.IO SHY.KCX TCG.KCXTCG.B4C Week 1 +++ −/+ ++ ++ Week 2 +++ −/+ +++ +++ Week 3 +++ ++ ++++++ Week 4 +++ ++ +++ +++ Key: − no growth; −/+ poor growth; + fairgrowth; ++ good gowth; +++ very good growth

TABLE 37 Salinipora tropica NPS21184, 2^(nd) streak SHY.IO SHY.KCXTCG.KCX TCG.B4C Week 1 +++ + ++ ++ Week 2 +++ ++ +++ +++ Week 3 +++ +++++ +++ Week 4 +++ ++ +++ +++ Key: − no growth; −/+ poor growth; + fairgrowth; ++ good gowth; +++ very good growth

Good growth of all three Salinispora tropica strains was observed onTCG.KCX agar media after only one week of incubation. The growth rate ofthree Salinispora tropica strains on the low sodium (11 mM), non-salineTCG.KCX agar medium was similar to the high sodium (452 mM), saline agarmedia (SHY.IO and TCG.B4C) at the second streak. Growth of theSalinispora tropica strains was also observed in the other low sodium,non-saline SHY.KCX agar but the growth yield was not as good as theTCG.KCX agar medium.

Example 14 Effect of Vitamin BI on Production of Salinosporamide A.NPI-0047 and NPI-2065 by Salinispora tropica NPS21184 in the INSTANTOCEAN® Commercial Salt Formulation

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into four different seed media (Instant Ocean® based)with the following compositions (per liter of DI H₂O).

Medium 0x 1x 4x 10x Components Vitamin B₁₂ Vitamin B₁₂ Vitamin B₁₂Vitamin B₁₂ Starch 10 g 10 g 10 g 10 g Hy Soy 4 g 4 g 4 g 4 g YeastExtract 4 g 4 g 4 g 4 g KBr 100 mg 100 mg 100 mg 100 mg Fe₂SO₄ 40 mg 40mg 40 mg 40 mg CaCO₃ 100 mg 100 mg 100 mg 100 m INSTANT 30 g 30 g 30 g30 g OCEAN ® Vitamin B₁₂ 0 0.296 mg 1.185 mg 2.96 mg (0.22 μM) (0.88 μM)(2.2 μM)

After incubating the above first seed cultures (10 ml medium in a 50-mltube) for 72 hours at 250 rpm and 28° C., 5 mL of each first seedculture was inoculated into 100 mL of the second seed medium (in 500-mLErlenmeyer flask) having the same composition as the first seed medium.The second seed cultures were incubated at 250 rpm and 28° C. for 48hours. 5 mL of each of the second seed cultures was inoculated into 100mL of the production medium (in 500 mL Erlenmeyer flask) containing thesame composition as the seed medium. The 0× Vitamin B₁₂ second seedculture (no vitamin B₁₂ in seed medium) was also inoculated intoadditional production medium containing 0.296 mg/L Vitamin B₁₂ (lxvitamin B₁₂P).

About 24 hour after inoculation, sterile XAD-7 resin slurry was added tothe production cultures in a final concentration of ˜20 g/L. Theproduction culture was then further incubated and harvested at varioustime points to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and an isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min for 15min with the detector wavelength set at 210 nm and column temperature35° C.

TABLE 38 Maximum production of Salinosporamide A, NPI-0047 and NPI-2065by Salinispora tropica NPS21184 in production media (Instant Ocean ®based) supplemented with vitamin B₁₂ [Vitamin B₁₂] [Vitamin B₁₂] MaximumTiter (mg/L) Media in Seed in Production Salinosporamide conditionsMedium Medium A NPI-0047 NPI-2065 0x Vitamin 0 0 252 11.1 6.3 B₁₂ 1xvitamin 0 0.296 mg/L 231 2.9 4.4 B₁₂P (0.22 μM) 1x Vitamin 0.296 mg/L0.296 mg/L 266 2.8 4.9 B₁₂ (0.22 μM) (0.22 μM) 4x Vitamin 1.185 mg/L1.185 mg/L 266 2.8 5.4 B₁₂ (0.88 μM) (0.88 μM) 10x Vitamin  2.96 mg/L 2.96 mg/L 247 2.5 4.9 B₁₂ (2.2 μM) (2.2 μM)

Vitamin B₁₂, like cobalt, significantly reduced the concentration ofNPI-0047 in the fermentation. Adding 0.22 μM of vitamin B₁₂ to theproduction medium (Instant Ocean® based) effectively reduced theconcentration of NPI-0047 by 74%. Including vitamin B₁₂ in the seedmedium or increasing the vitamin B₁₂ concentration in both seed andproduction media by 10 fold (2.2 μM) further reduced the concentrationof NPI-0047 only slightly. Vitamin B₁₂ also reduced the concentration ofNPI-2065 by 30% in the Instant Ocean® based medium.

Example 15 Effect of Vitamin B₁₂ and Cobalt on Production ofSalinosporamide A. NPI-0047 and NPI-2065 by Salinispora tropica NPS21184in the Salt Formulation Based Medium

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into three different seed media of varying saltcontent with the following compositions (per liter of DI H₂O).

Medium Components Control 1× Vitamin B₁₂ 1× CoCl₂•6H₂O Starch 10 g 10 g10 g Hy Soy 4 g 4 g 4 g Yeast Extract 4 g 4 g 4 g KBr 100 mg 100 mg 100mg Fe₂SO₄ 40 mg 40 mg 40 mg CaCO₃ 100 mg 100 mg 100 mg Vitamin B₁₂ 00.296 mg 0 (0.22 μM) 0 CoCl₂•6H₂O 0 0 52 μg (0.22 μM)

The seed medium was then supplemented with a chemically defined saltformulation. The chemically defined salt formulation was:

Salt component Per liter of DI H₂O NaCl 24 g KCl 0.69 g MgSO₄•7H₂O 4.29g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mg

After incubating the above first seed cultures (10 ml medium in a 50-mltube) for 72 hours at 250 rpm and 28° C., 5 mL of each first seedculture was inoculated into 100 mL of the second seed medium (in 500-mLErlenmeyer flask) having the same composition as the first seed medium.The second seed cultures were incubated at 250 rpm and 28° C. for 48hours. 5 mL of each of the second seed cultures was inoculated into 100mL of the production medium (in 500 mL Erlenmeyer flask) containing thesame composition as the seed medium.

About 24 hour after inoculation, sterile XAD-7 resin slurry was added tothe production cultures in a final concentration of ˜20 g/L. Theproduction culture was then further incubated and harvested at varioustime points to determine productivity.

Three and one-half (3.5) mL of the fermentation culture was mixed withequal volume of EtOAc in an extraction tube to extract the variousmetabolites from the production culture. The mixture was shaken on theshaker for 1 hr. An aliquot of the extract (1 mL) was removed and driedunder a stream of nitrogen. The dried extract was stored at −20° C.freezer before HPLC analysis. The dried extract was resuspended in 320μL DMSO and injected into HPLC using the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

TABLE 39 Maximum production of Salinosporamide A, NPI-0047 and NM-2065by Salinispora tropica NPS21184 in production media (defined saltformulation based) supplemented with cobalt or vitamin B₁₂ Maximum Titer(mg/L) Media Seed Production Salinosporamide NPI- NPI- conditions MediumMedium A 0047 2065 Control No vitamin No vitamin B₁₂ 277 4.7 5.6 B₁₂ orcobalt or cobalt supplement supplement 1× vitamin B₁₂ 0.296 mg/L 0.296mg/L 304 1.1 7.2 (0.22 μM) (0.22 μM) Vitamin B₁₂ Vitamin B₁₂ 1×CoCl₂•6H₂O 52 μ/L 52 μ/L, 297 1.4 6.6 (0.22 μM) (0.22 μM) CoCl₂•6H₂OCoCl₂•6H₂O

Vitamin B₁₂ and CoCl₂.6H₂O, at equal concentration (0.22 μM), have thesame effect in reducing the concentration of NPI-0047 by 70 to 77% inthe salt formulation based medium. There is no reduction inconcentration of NPI-2065 from Vitamin B₁₂ or CoCl₂.6H₂O in the saltformulation based medium

Example 16 Production of NPI-0052 in Low Chloride (19 mM), SodiumSulfate Based-Medium in Fermentor Culture

Seed cultures were prepared by inoculating frozen stock of Salinisporatropica NPS021184 into seed medium consisting the following per liter ofDI H₂O:

10 g Starch 4 g Hy Soy 4 g Yeast Extract 1 g CaCO₃ 100 mg KBr 40 mgFe₂(SO₄)₃

The seed medium was then supplemented with a chemically defined saltformulation. The chemically defined salt formulation included:

Salt component Per liter of DI H₂O Na₂SO₄ 40 g KCl 0.69 g MgSO₄•7H₂O4.29 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mgCoCl₂•6H₂O 208 μg

The first seed culture (100 mL medium) in a 500-mL Erlenmeyer flask wasincubated at 250 rpm and 28° C. for 72 hours. 20 mL of the first seedculture was inoculated into second seed culture (400 ml medium) in a 2.8L Fernbach flask with the same medium as the first seed culture. Thesecond seed culture was incubated at 250 rpm and 28° C. for 48 hours.200 mL of the third seed culture was inoculated into a B. Braun B-Plus 5L fermentor containing 4 L third seed medium with the same compositionas the first and second seed medium. The thirds seed fermentor culturewas incubated at 300 rpm and 28° C. with the airflow rate of 2 L/min for36 hours. 1.5 L of the third seed culture was inoculated into 26 Lproduction medium in a 42 L B. Braun Biostat-C fermentor.

Per liter of DI H₂O, the production medium consisted of:

10 g Starch 4 g Hy Soy 4 g Yeast Extract 1 g CaCO₃ 100 mg KBr 40 mgFe₂(SO₄)₃

The production medium was then supplemented with a chemically definedsalt formulation consisted of:

Salt component Per liter of DI H₂O Na₂SO₄ 20 g KCl 0.69 g CaCl₂•2H₂O0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208 μg

The ion content of the production medium was analyzed by ICP-MS analysisand found to have the following composition:

TABLE 40 ICP-MS analysis of ions (mM) present in the medium [Na] [Cl][K] [Mg] [Co] [S] [Ca] [Fe] 284 19 14 0.56 1.1 × 10⁻³ 159 3.8 0.16

The chloride content in the production medium is 19 mM, which issignificantly F lower than the chloride content in the INSTANT OCEAN®based medium (424 mM) and the defined salt formulation based medium (426mM) (Table 20 of Example 9). The production fermentor culture wasoperated at 200 rpm and 28° C. with the airflow rate of 13 L/min. About39 hours after inoculation, sterile XAD-7 resin slurry was added to theproduction culture in a final concentration of 20 g/L. The productionculture was then further incubated and harvested at various time pointsto determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

TABLE 41 The production time course of NPI-0052, NPI-0047 and NPI-2065in the low chloride medium by Salinispora tropica NPS21184 in the 42 Lfermentor Titers (mg/L) Culture Salinosporamide Age (hours) A NPI-0047NPI-2065 74 77 3.7 2.4 83 85 3.6 2.6 98 164 7.7 4.6 106 198 12.1 5.2 123225 15.5 6.1 130 229 15.6 6.4

Sodium sulfate effectively replaced sodium chloride in supporting theproduction of NPI-0052 in fermentor culture. The sulfate medium contains19 mM chloride concentration that supported the production of 229 mg/LNPI-0052 in fermentor culture. The major chloride in the medium wasderived from KCl, CaCl₂ and carried over from Hy Soy and Yeast Extract.

Example 17 Comparison of Growth of Low Sodium Frozen Stocks ofSalinispora tropica CNB440, CNB476 and NPS021184 Grown in Low SodiumMedium with Omission of NaF (No Discrete Sodium Salt Added)

Low Sodium Frozen Stocks of Salinispora tropica CNB440, CNB476 andNPS021184 prepared in Example 10 were used to inoculate 10 mL aliquotsof the SHY.KCK seed medium in 50-mL culture tubes (25×150 mm).

SHY.KCK (per liter of DI H₂O)

10 g Starch 4 g Hy Soy 4 g Yeast Extract 40 mg Fe₂(SO₄)₃ 30 g KCl 4.29 gMgSO₄.7H₂O 0.43 g CaCl₂.2H₂O 15.5 mg SrCl₂ 21.5 mg H₃BO₃

2.6 mg/L KF

208 μg CoCl₂.6H₂O

No discrete sodium salt was included in or added to Medium SHY.KCK.SHY.KCK differs from SHY.KCD in the omission of 2.6 mg/L NaF and 1 g/LCaCO₃ from SHY.KCD and the addition of 2.6 mg/L of KF in the medium.Therefore, Medium SHY.KCK contains no known addition of sodium salt. Thesodium present in the medium is derived from other medium components.The ion content of Medium SHY.KCK was analyzed by ICP-MS analysis andfound to have the following composition:

TABLE 42 ICP-MS analysis of ions (mM) present in the medium [Na] [Cl][K] [Mg] [Co] [S] [Ca] [Fe] 10 482 407 24 1.2 × 10⁻³ 22 3.7 0.17

1 mL of the frozen stock each was used to inoculate 10 mL first seedSHY.KCK medium to yield an inoculum size of 10%. After incubating theabove first seed cultures of CNB476 and NPS21184 for 48 hours at 250 rpmand 28° C., 5 mL of each culture was inoculated into 100 mL of thesecond seed medium (in 500-mL Erlenmeyer flask) having the samecomposition as the first seed medium. For CNB440, the first seed culturewas grown for 5 days. 10 mL of the first seed culture was used toinoculate 100 mL of the second seed medium in a 500-mL Erlenmeyer flask.The dry cell weights of the first seed cultures at the time ofinoculation to the second seed medium are shown in the following table:

TABLE 43 The dry cell weight of the first seed cultures of strainsCNB440, CNB476 and NPS21184 grown in Medium SHY•KCK Strain Culture age(hours) Dry cell weight (mg/mL) CNB440 120 1.9 CN476 48 3.8 NPS21184 484.5

Good cell growth of strains CNB476 and NPS21184 was observed after 48hours incubation in the first seed culture in medium SHY.KCK. The growthrate and cell yield of strain CNB440 are lower than strains CNB476 andNPS21184, yielding a dry cell weight of 1.9 mg/L after 120 hourincubation in first seed culture growing in Medium SHY.KCK. The secondseed cultures were incubated for 24 to 120 hours and yielded thefollowing dry cell weight data:

TABLE 44 The dry cell weight of the first seed cultures of strainsCNB440, CNB476 and NPS21184 grown in Medium SHY•KCK Dry cell weight(mg/mL) Strain 24 hours 48 hours 72 hours 96 hours 120 hours CNB440 ND0.92 2.1 3.1 3.3 CNB476 0.48 2.8 3.4 3.5 3.8 NPS21184 0.97 2.4 3.1 3.33.8 ND = Not determined

The dry cell weight data from the second seed cultures confirmed thefinding that the Low Sodium Frozen Stocks of CNB440, CNB476 and NPS21184prepared in the low sodium medium (9.3 mM) can support good cell growthin low sodium Medium SHY.KCK which includes added trace metal ions otherthan sodium. The Low Sodium Frozen of strains CNB440, CNB476 andNPS21184 grew very well in the low sodium medium (10 mM sodium) with drycell weights of 3.3 to 3.8 mg/mL. While the growth rate of CNB440 isslower in the low sodium medium, it achieved the same dry cell weight asNPS21184 after 96 hours incubation. Since Medium SHY.KCK contains 2.6mg/L of KF, KF or fluoride ion may play a role in supporting the growthof Salinispora tropica CNB440, CNB476 and NPS21184.

Example 18 Growth of Salinispora tropica CNB440 and CNB476 andProduction of Salinosporamide A in Non-Saline, Low Sodium, Seed andProduction Media Containing Trace Amount of Sodium Ion (CalculatedValue: 0.06 mM; ICP-MS Analysis: 11 mM)

The first seed cultures were prepared by inoculating two frozen stockseach of Salinispora tropica CNB440 and CNB476 into first seed mediumcontaining INSTANT OCEAN® commercial salt formulation to obtain goodgrowth of the culture. The first seed culture was centrifuged and washedwith Wash Media containing no addition of sodium salt to remove thesodium ion present in the first seed culture prior to inoculation intothe second seed stage containing medium with only trace amounts ofsodium ion (calculated value of sodium derived from 2.6 mg/L NaF: 0.06mM, while no ICP-MS analysis was performed, it is estimated that thesodium content is ˜5.7 mM based on ICP-MS analysis of a similar mediumA1.Ks4C, Table 14A of Example 7). The third seed culture was theninoculated into the production medium SHY.KcMC containing trace amountof sodium ion (calculated value of sodium derived from 2.6 mg/L NaF:0.06 mM; ICP-MS analysis: 11 mM, Table 15 of Example 8). This processsignificantly reduced the carried over of sodium ion from the seedcultures to the production culture.

The first seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then supplemented with 30 g/L INSTANT OCEAN® commercial saltformulation.

The first seed culture (100 ml medium containing ˜424 mM sodium ion fromInstant Ocean) in a 500-mL Erlenmeyer flask was incubated at 28° C. and250 rpm for 72 hours. 10 mL of the first seed culture was transferred toa sterile 15-mL centrifuge tube and centrifuged at 3,000 rpm for 15 min.The packed cell volume observed was 5% for both strain 429 and strain465. The supernatant was decanted and 10 mL of Wash Medium (10 g/Lstarch, 2 g/L peptone, 4 g/L yeast extract, and 30 g/L KCl in D.I.water) was added to the centrifuge tube. The centrifuge tube was mixedand then centrifuged at 3000 rpm for 15 min. The supernatant wasdecanted and the cell culture was washed again by re-suspending in WashMedia and centrifuging and decanting as before. Then 10 mL of the secondseed medium (containing 0.06 mM sodium ion from NaF) was added to thecentrifuge tube. The second seed medium consisted of starch (USB,catalog #21695), peptone (USB, catalog #20048), and yeast extract (USB,catalog #23547) at concentrations of 10 g/L, 2 g/L, and 4 g/L,respectively. The seed medium was also supplemented with a chemicallydefined salt formulation. This supplemented medium is referred to asA1.Kc4C. The chemically defined salt formulation consisted of:

Salt component Per liter of DI H₂O KCl 30 g MgSO₄•7H₂O 4.29 g KBr 85.9mg CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mgCoCl₂•6H₂O 208 μg

The centrifuge tube was gently vortexed to mix the cell suspension and 5mL of the washed cells were inoculated to the second seed culturecontaining 100 ml medium having the same composition as the second seedmedium in a 500-mL Erlenmeyer flask. The sodium concentration in thewashed cell inoculum had been reduced down to a calculated level of˜0.11 mM sodium based on 5% packed cell volume, which is double theamount of sodium content in the second seed medium (0.06 mM from 2.6mg/L NaF). The second seed culture was incubated for 6 days beforeinoculating 5 mL of the second seed culture into the third seed culturecontaining the same medium as the second seed stage. With furtherdilution of the second seed inoculum by the medium of the third seedstage, the sodium content in the third seed culture is essentiallycalculated as ˜0.06 mM (contributed by 2.6 mg/L NaF). The third seedcultures of CNB440 and CNB476 were incubated for 5 days and 3 days,respectively, before inoculating 5 ml of the third seed culture into theproduction medium (100 ml in a 500-ml Erlenmeyer flask) with the samecomposition of the defined salt supplemented-production medium,SHY.KcMC, in Example 5.

Per liter of DI H₂O, the production medium SHY.KcMC consisted of:

10 g Starch

4 g Hy Soy

4 g Yeast Extract

1 g CaCO₃

100 mg KBr

The production medium was then supplemented with a chemically definedsalt formulation. The chemically defined salt formulation consisted of:

Salt component Per liter of DI H₂O KCl 30 g CaCl₂•2H₂O 0.43 g SrCl₂ 15.5mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52 μg

About 24 after inoculation, sterile XAD-7 resin slurry was added to theproduction cultures in a final concentration of 20 g/L. The productionculture was then further incubated and harvested at various time pointsto determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and an isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min for 15min with the detector wavelength set at 210 nm and column temperature35° C.

TABLE 45 Production of Salinosporamide A, NPI-0047 and NPI-2065 bySalinispora tropica CNB440 grown in production medium containing 0.06 mMsodium ion based on NaF present in medium and 11 mM sodium ion based onICP-MS analysis. Titer (mg/L) Culture Age (days) Salinosporamide ANPI-0047 NPI-2065 3 0 0 0 4 0 0 0 5 28 0.4 0.3 6 67 0.6 0.6

TABLE 46 Production of Salinosporamide A, NPI-0047 and NPI-2065 bySalinispora tropica CNB476 grown in production medium containing 0.06 mMsodium ion based on NaF present in medium and 11 mM sodium ion based onICP-MS analysis. Titers (mg/L) Culture Age (days) Salinosporamide ANPI-0047 NPI-2065 3 0 0 0 4 64 0.2 1.0 5 107 0.8 0.3 6 147 0.1 1.0

Salinispora tropica CNB440 and CNB476 can be grown in production mediumwith a low sodium ion concentration of 11 mM, which is about 2% of thesodium content in sea water (487 mM sodium, see Table 20 of Example 9).In this low sodium production medium, Salinispora tropica CNB440 andSalinispora tropica CNB476 produced 67 mg/L and 147 mg/L ofSalinosporamide A, respectively. Since Salinispora tropica CNB440 andSalinispora tropica CNB476 can be grown in the A1.Kc4C seed media withsodium content estimated at 5.7 mM by ICP-MS analysis, this demonstratedthat Salinispora tropica CNB440 and Salinispora tropica CNB476 can begrown in medium with sodium content at least as low as 1% of seawater.

Example 19 Growth of Salinispora tropica CNB440 and CNB476 in Agar MediaContaining (a) Low Sodium and (b) Low Sodium and Low Chloride

The first seed culture was prepared by inoculating two frozen stockseach of Salinispora tropica CNB440 and CNB476 into first seed mediumconsisted of glucose, Hy Soy, and yeast extract at concentrations of 8g/L, 6 g/L, and 6 g/L, respectively. The seed medium was thensupplemented with 30 g/L INSTANT OCEAN® commercial salt formulation.

The first seed culture (100 mL medium containing ˜424 mM sodium ion fromINSTANT OCEAN®) in a 500-mL Erlenmeyer flask was incubated at 28° C. and250 rpm for 72 hours. 1.5 mL of the first seed culture each wastransferred to two sterile 15-mL centrifuge tubes and centrifuged at3,000 rpm for 15 min. The supernatant from each tube was decanted. 10 mLof Wash Medium A was added to one tube while 10 mL of Wash Medium B wasadded to the other tube. Both wash media consisted of 10 g/L starch, 2g/L peptone, 4 g/L yeast extract and either 30 g/L KCl (Wash Media A) or48 g/L K₂SO₄ (Wash Media B). The centrifuge tubes were mixed and thencentrifuged at 3000 rpm for 15 min. The supernatant was decanted and thecells were resuspended in the same medium and centrifuged again. Afterthe final centrifuging, a pipette was used to remove most of thesupernatant and allow enough remaining medium to yield a 2 ml cellsuspension in the tube. These cell suspensions were used as the inoculumfor the agar cultures. Four agar media were used and all consisted ofstarch, peptone, yeast extract, and agar (Difco, catalog #214530) atconcentrations of 10 g/L, 2 g/L, 4 g/L and 17 g/L, respectively, plus asalt supplement. Agar Medium A used the following chemically definedsalt supplement. This supplemented medium is referred to as A1.Kc4C

Agar Medium A—Salt Supplement Salt component Per liter of DI H₂O KCl 30g MgSO₄•7H₂O 4.29 g KBr 85.9 mg CaCO₃ 0.43 g CaCl₂•2H₂O 429.3 mg SrCl₂15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208 μg

In order to test the significance of the ion supplements other thanpotassium, Agar Medium A-1 used only 30 g/L KCl as the salt supplement.That is, to examine if simple replacement of NaCl by KCl in the agarmedium can support the growth of Salinispora tropica CNB440 and CNB476.

The Agar Medium B used the following chemically defined salt supplement.This supplemented medium is referred to as A1.Ks4C.

Agar Medium B—Salt Supplement Salt component Per liter of DI H₂O K₂SO₄48 g MgSO₄•7H₂O 4.29 g KBr 85.9 mg CaCO₃ 0.43 g CaCl₂•2H₂O 429.3 mgSrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 208 μg

In order to test the significance of the ion supplements other thanpotassium, Agar Medium B-1 used only 48 g/L K₂SO₄ as its supplement.That is, to examine if simple replacement of NaCl by K₂SO₄ in the agarmedium can support the growth of Salinispora tropica CNB440 and CNB476.Particular ion concentration of Agar Medium B with the complete saltsupplements (Medium A1.Ks4C) were determined by ICP-MS (Table 14A,Example 7).

Sterile inoculation loops were used to transfer cells from Wash Medium Ato agar plates containing 20 mL of Agar Medium A (calculated sodiumcontent: 0.06 mM; calculated chloride content: 415 mM) and 20 mL of AgarMedium A-1 (calculated sodium content: 0.06 mM; calculated chloridecontent: 403 mM). Sterile inoculation loops were used to transfer cellsfrom Wash Medium B to agar plates containing 20 mL of Agar Medium B(calculated sodium content: 0.06 mM; calculated chloride content: 12 mM;ICP-MS analysis: sodium content: 5.7 mM; chloride content: 14 mM) and 20mL of Agar Medium B-1 (calculated sodium content: 0.06 mM; calculatedchloride content: ˜0). After inoculation, the edges of the agar plateswere wrapped with parafilm to avoid evaporation. The agar plates wereincubated at 28° C. for 2-4 weeks to observe good growth. No growth wasobserved in Agar Medium A-1 or Agar Medium B-1 after 4 weeks incubation.Good growth was observed in Agar Medium A and Medium B after two weeksof incubation. After two weeks of incubation, the growth from AgarMedium A and Agar Medium B were transferred to fresh Agar Medium A andAgar Medium B plates, respectively. The re-streaked agar plates wereagain incubated at 28° C. to observe growth. The observation of growthof Salinispora tropica CNB440 and Salinispora tropica CNB476 on thesere-streaked agar plates were summarized in the following tables.

TABLE 47 Observation of growth of Salinispora tropica CNB440 grown onagar medium A1.Kc4C and A1.Ks4c (second streaked) Agar Medium Week 1Week 2 Week 3 Week 4 A (A1.Kc4C) ++ ++ +++ +++ B (A1.Ks4C) +++ +++ ++++++ Key: − no growth; −/+ poor growth; + fair growth; ++ growth; +++very good growth

TABLE 48 Observation of growth of Salinispora tropica CNB476 grown onagar medium A1.Kc4C and A1.Ks4C (second streaked) Agar Medium Week 1Week 2 Week 3 Week 4 A (A1.Kc4C) +++ +++ +++ +++ B (A1.Ks4C) ++ ++ ++++++ Key: − no growth; −/+ poor growth; + fair growth; ++ growth; +++very good growth

Both Salinispora tropica strains CNB440 and CNB476 can be grown in agarmedia containing either low sodium (5.7 mM) and low chloride (14 mM),providing that the agar media is also supplemented with other ions suchas MgSO₄, KBr, CaCO₃, CaCl₂, SrCl₂, H₃BO₃, NaF and CoCl₂. Substitutionof KCl or K₂SO₄ for NaCl alone cannot support the growth of Salinisporatropica strains NPS429 and NPS465 in agar media.

Example 20 The Effect of Sodium Chloride in the Production Medium on theProduction of Salinosporamide A, NPI-0047 and NPI-2065 by Salinisporatropica CNB440, CNB476 and NPS21184

Seed cultures were prepared by inoculating frozen stocks of Salinisporatropica CNB440, CNB476 and NPS21184 into seed medium.

The seed medium consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediumwas then supplemented with a chemically defined salt formulationconsisted of:

Salt component Per liter of DI H₂O NaCl 24 g KCl 0.69 g MgSO₄•7H₂O 4.29g KBr 85.9 mg CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mgNaF 2.6 mg CoCl₂•6H₂O 208 μg

The first seed cultures (100 mL medium) in 500-mL Erlenmeyer flasks wereincubated for 96 hours before inoculating 5 mL of the first seed cultureinto the second seed medium with the same composition as the first seedmedium. The second seed cultures (100 mL medium in 500-mL Erlenmeyerflask) were incubated for 48 hours before inoculating 5 mL of the secondseed culture into the production medium (100 mL in a 500-mL Erlenmeyerflask).

Per liter of DI H2O, the production medium consisted of: 10 g Starch, 4g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, and 100 mg KBr.The production medium was then supplemented with a chemically definedsalt formulation consisted of the following common salt components alongwith different levels of NaCl:

Salt component Per liter of DI H₂O KCl 0.69 g CaCl₂•2H₂O 0.43 g SrCl₂ 155 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52 μg

The NaCl levels used were 0, 5, 10, 15, 20, 24 and 30 g/L. About 24after inoculation, sterile XAD-7 resin slurry was added to theproduction cultures in a final concentration of 20 g/L. The productionculture was then further incubated and harvested at various time pointsto determine productivity.

Three and one-half (3.5) mL of the fermentation culture was mixed withequal volume of EtOAc in an extraction tube to extract the variousmetabolites from the production culture. The mixture was shaken on theshaker for 1 hr. An aliquot of the extract (1 mL) was removed and driedunder a stream of nitrogen. The dried extract was stored at −20° C.freezer before HPLC analysis. The dried extract was resuspended in 320μL DMSO and injected into HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and an isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min with thedetector wavelength set at 210 nm and column temperature 35° C.

TABLE 49 Production of Salinosporamide A by Salinispora tropica CNB440,CNB476, and NPS021184 grown in production medium containing saltformulation with different concentrations of NaCl. NaCl ConcentrationTiter (mg/L) (g/L) CNB440 CNB476 NPS21184 0 0 0 0 5 19 0 5.4 10 55 72235 15 63 96 288 20 83 111 286 24 84 116 295 30 97 115 223

TABLE 50 Production of NPI-0047 by Salinispora tropica CNB440, CNB476,and NPS021184 grown in production medium containing salt formulationwith different concentrations of NaCl. NaCl Concentration Titer (mg/L)(g/L) CNB440 CNB476 NPS21184 0 0 0 0 5 0.8 0 0.5 10 4.6 4.9 14 15 4.55.4 6.4 20 5.2 3.2 1.2 24 4.8 2.9 2.1 30 3.2 1.3 1.4

TABLE 51 Production of NP1-2065 by Salinispora tropica CNB440, CNB476,and NPS021184 grown in production medium containing salt formulationwith different concentrations of NaCl. NaCl Concentration Titer (mg/L)(g/L) CNB440 CNB476 NPS21184 0 0 0 0 5 0.4 0 0 10 0.9 1.0 3.9 15 0.9 0.56.2 20 0.4 1.1 6.2 24 0.5 1.1 6.2 30 0.6 1.1 4.2

The growth yield of the day 6 production cultures of Salinispora tropicaCNB440, CNB476, and NPS021184 grown in production medium containing saltformulation with different concentrations of NaCl was determined bypacked cell volume and is shown the following table.

TABLE 52 The growth yield of the day 6 production cultures ofSalinispora tropica CNB440, CNB476, and NPS021184 grown in productionmedium containing salt formulation with different concentrations ofNaCl. NaCl Concentration Packed Cell Volume (%) (g/L) CNB440 CNB476NPS21184 0 0 0 0 5 5 2 6 10 5 5 6 15 4 5 6 20 5 6 5 24 5 6 5 30 5 6 5

No growth was observed for any of the three strains when the aboveproduction medium was not supplemented with NaCl. Both Salinisporatropica CNB440 and NPS21184 achieved good cell yield when the aboveproduction medium was supplemented with 5 g/L NaCl. There was nodifference in the cell yield with further increase in the concentrationof NaCl from 10 g/L to 30 g/L. Salinispora tropica CNB476 requiredhigher concentration of NaCl, 10 g/L, for achieving good growth yield.

The production of NPI-0052 was detected, at low concentration, in bothSalinispora tropica CNB440 (19 mg/L) and NPS21184 (5.4 mg/L) in themedium supplemented with 5 g/L NaCl. No NPI-0052 was detected inSalinispora tropica CNB476 grown in 5 g/L NaCl and this may be relatedto the poor cell yield. Salinispora tropica NPS21184 is clearly the bestNPI-0052-producer among the three strains with the maximum titer of 295mg/L detected at 24 g/L NaCl while Salinispora tropica CNB440 and CNB476had maximum titers of 97 mg/L and 116 mg/L, respectively.

For the production of NPI-0074, there is a trend of decreasingconcentration of NPI-0047 with increasing concentration of NaCl in theproduction medium. The production of NPI-0047 is the lowest inSalinispora tropica NPS21184, among the three strains tested. Theproduction of NPI-2065 is the highest in Salinispora tropica NPS21184,among the three strains tested.

Example 21 Compare the Effect of Cobalt on the Production ofSalinosporamide A, NPI-0047 and NPI-2065 by Salinispora tropica CNB440,CNB476 and NPS21184

Seed cultures were prepared by inoculating frozen stocks of Salinisporatropica CNB440, CNB476 and NPS21184 into two different seed media.

Both seed media consisted of glucose, Hy Soy, and yeast extract atconcentrations of 8 g/L, 6 g/L, and 6 g/L, respectively. The seed mediawere then supplemented with a chemically defined salt formulationconsisting of:

Salt component Per liter of H₂O NaCl 24 g KCl 0.69 g MgSO₄•7H₂O 4.29 gKBr 85.9 mg CaCO₃ 0.43 g CaCl₂•2H₂O 0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mgNaF 2.6 mg

One seed medium was further supplemented with 208 μg/L CoCl₂.6H₂O. Thefirst seed cultures (100 mL medium) in 500-mL Erlenmeyer flasks wereincubated for 72 hours before inoculating 5 mL of the first seed cultureinto the second seed medium with the same composition as the first seedmedium. The second seed cultures (100 mL medium in 500-mL Erlenmeyerflask) were incubated for 48 hours before inoculating 5 mL of the secondseed culture into the production media (100 mL in a 500-mL Erlenmeyerflask) containing the following common ingredient, per liter of DI H₂O:10 g Starch, 4 g Hy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃,and 100 mg KBr. The production media was then supplemented with achemically defined salt formulation consisting of the following commonsalt components:

Salt component Per liter of DI H₂O NaCl 24 g/L KCl 0.69 g CaCl₂•2H₂O0.43 g SrCl₂ 15 5 mg H₃BO₃ 21.5 mg NaF 2.6 mg

One production medium was also supplemented with 52 g/L CoCl₂.6H₂O. Theseed culture with no cobalt was inoculated into the production mediumwith no added cobalt. The seed culture containing 208 g/L CoCl₂.6H₂O wasinoculated into production medium with 52 μg/L CoCl₂.6H₂O. After about24 hour incubation into the production culture, sterile XAD-7 resinslurry was added to the production cultures in a final concentration of20 g/L. The production culture was then further incubated and harvestedat various time points to determine productivity.

Three and one-half (3.5) mL of the fermentation culture was mixed withequal volume of EtOAc in an extraction tube to extract the variousmetabolites from the production culture. The mixture was shaken on theshaker for 1 hr. An aliquot of the extract (1 mL) was removed and driedunder a stream of nitrogen. The dried extract was stored at −20° C.freezer before HPLC analysis. The dried extract was resuspended in 320μL DMSO and injected into HPLC using the TFS7 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and an isocratic solvent system consisting of 67% water (0.01% TFA)and 33% acetonitrile (0.01% TFA). The flow rate was 1.5 mL/min with thedetector wavelength set at 210 nm and column temperature 35° C.

TABLE 53 Production of Salinosporamide A by Salinispora tropica CNB440,CNB476, and NPS021184 grown in seed and production medium containingdifferent concentrations of cobalt. [CoCl₂•6H₂O] [CoCl₂•6H₂O] inproduction Titer (mg/L) in seed medium medium CNB440 CNB476 NPS21184 0 0100 111 258 208 μg/L 52 μg/L  84 111 292

TABLE 54 Production of NPI-0047 by Salinispora tropica CNB440, CNB476,and NPS021184 grown in seed and production medium containing differentconcentrations of cobalt. [CoCl₂•6H₂O] [CoCl₂•6H₂O] in production Titer(mg/L) in seed medium medium CNB440 CNB476 NPS21184 0 0 13 17 13 208μg/L 52 μg/L 6.8 8.1 1.2

TABLE 55 Production of NH-2065 bySalinispora tropica CNB440, CNB476, andNPS021184 grown in seed and production medium containing differentconcentrations of cobalt. [CoCl₂•6H₂O] [CoCl₂•6H₂O] in production Titer(mg/L) in seed medium medium CNB440 CNB476 NPS21184 0 0 0.78 1.3 6.5 208g/L 52 g/L 0.69 0.88 5.4

This example supports the theory that the addition of cobalt to the seedand production media reduced the production of NPI-0047 in all threeSalinispora tropica strains. However, the three Salinispora tropicastrains have different degree of reduction of NPI-0047 by cobalt.Salinispora tropica NPS21184 shows the highest percentage (91%) ofreduction of NPI-0047 by cobalt. Cobalt reduced the production ofNPI-0047 by Salinispora tropica CNB440 and CNB476 by 48% and 52%,respectively.

Example 22 Examination of the Effect of Cobalt in INSTANT OCEAN®Commercial Salt Formulation-Based Seed and Production Media on theProduction of Salinosporamide A, NPI-0047 and NPI-0065 by Salinisporatropica NPS21184

Seed cultures were prepared by inoculating frozen stocks of Salinisporatropica NPS21184 into seed medium. The seed medium consisted of glucose,Hy Soy, yeast extract and INSTANT OCEAN® at concentrations of 8 g/L, 6g/L, 6 g/L, and 30 g/L, respectively. The seed media were supplementedwith 0, 52 μg/L or 208 μg/L of CoCl₂.6H₂O. The first seed cultures (100mL medium in 500-mL Erlenmeyer flask) were incubated for 3 days beforeinoculating into the second seed media with the same composition of thefirst media. The second seed cultures (100 mL medium in 500-mLErlenmeyer flask) were incubated for 2 days before inoculating into theproduction media (100 mL in 500-mL Erlenmeyer flask).

Per liter of DI H₂O, the production media consisted of: 10 g Starch, 4 gHy Soy, 4 g Yeast Extract, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃, 100 mg KBr, and30 g INSTANT OCEAN® The production media were supplemented with 0, 13μg/L, 52 μg/L or 208 μg/L of CoCl₂.6H₂O. The following table summarizesthe conditions used for this study

Condition Seed medium Production medium 1 (Control) No cobalt additionNo cobalt addition 2 No cobalt addition +13 μg/L CoCl₂•6H₂O 3 No cobaltaddition +52 μg/L CoCl₂•6H₂O 4 No cobalt addition +208 μg/L CoCl₂•6H₂O 5+52 μg/L CoCl₂•6H₂O No cobalt addition 6 +52 μg/L CoCl₂•6H₂O +13 μg/LCoCl₂•6H₂O 7 +52 μg/L CoCl₂•6H₂O +52 μg/L CoCl₂•6H₂O 8 +52 μg/LCoCl₂•6H₂O +208 μg/L CoCl₂•6H₂O 9 +208 μg/L CoCl₂•6H₂O No cobaltaddition 10 +208 μg/L CoCl₂•6H₂O +13 μg/L CoCl₂•6H₂O 11 +208 μg/LCoCl₂•6H₂O +52 μg/L CoCl₂•6H₂O 12 +208 μg/L CoCl₂•6H₂O +208 μg/LCoCl₂•6H₂O

After 24 hours of inoculation into the production media, sterile XAD-7resin slurry was added to the production cultures in a finalconcentration of 20 g/L. The production cultures were then furtherincubated and harvested at various time points to determineproductivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the 00TF14 method for analysis.

The production of Salinosporamide A, NPI-0047 and NPI-2065 was monitoredby Agilent HP1100 HPLC using an ACE C-18 reversed-phase column (4.6×150mm), and solvent system consisting of water (0.01% TFA) as solvent A andacetonitrile (0.01% TFA) as solvent B. The elution was started at 100%solvent A for 1 min, 100% solvent A to 35% solvent A gradient in 7 min,held at 35% solvent A for 11 min, 35% solvent A to 100% solvent B in 8min and held at 100% solvent B for 9 min at a flow rate of 1.5 mL/minwith the detector wavelength set at 210 nm and column temperature 35° C.

The highest Salinosporamide A titer was detected at day 4 of theproduction cycle. The titers of Salinosporamide A, NPI-0047 and NPI-2065at day 4 of the production cycle are summarized in the following table.

TABLE 56 Production of Salinosporamide A, NPI-0047 and NPI-2065 inINSTANT OCEAN ®-based seed and production media supplemented withvarious concentrations of cobalt Seed Production Titers (mg/L)Conditions medium medium Salinosporamide A NPI-0047 NPI-2065 1 (Control)No cobalt No cobalt 275 15 7.3 addition addition 2 No cobalt +13 μg/L275 7.4 6.9 addition CoCl₂•6H₂O 3 No cobalt +52 μg/L 244 5.6 5.7addition CoCl₂•6H₂O 4 No cobalt +208 μg/L 244 5.5 6.0 additionCoCl₂•6H₂O 5 +52 μg/L No cobalt 281 11.8 6.2 CoCl₂•6H₂O addition 6 +52μg/L +13 μg/L 261 8.9 5.8 CoCl₂•6H₂O CoCl₂•6H₂O 7 +52 μg/L +52 μg/L 2527.8 5.7 CoCl₂•6H₂O CoCl₂•6H₂O 8 +5 μg/L +208 μg/L 256 7.0 5.9 CoCl₂•6H₂OCoCl₂•6H₂O 9 +208 μg/L No cobalt 257 7.9 6.4 CoCl₂•6H₂O addition 10 +208μg/L +13 μg/L 256 5.7 6.1 CoCl₂•6H₂O CoCl₂•6H₂O 11 +208 μg/L +52 μg/L265 6.4 6.5 CoCl₂•6H₂O CoCl₂•6H₂O 12 +208 μg/L +208 μg/L 246 5.1 6.1CoCl₂•6H₂O CoCl₂•6H₂O

The above data again supported the role of cobalt in reducing theproduction of NPI-0047 in Salinispora tropica. The above data suggestedthat cobalt present in the production media has a more significanteffect in reducing the production of NPI-0047.

Example 23 Improved the Production of NPI-2080 by Salinispora tropicaNPS21184 in a Non-Saline, Low Chloride Medium

In U.S. patent application Ser. No. 11/517,899, entitled “Biosynthesesof Salinosporamide A and its Analogs and Related Methods of MakingSalinosporamide A and its Analogs,” filed Sep. 8, 2006, which isincorporated herein by reference in its entirety, production of NPI-2080(II-30), a minor salinosporamide analog present in the salinefermentation of Salinispora tropica NPS21184, by feeding the culturewith valeric acid in Instant Ocean® based medium (Example 64, Table 25)was reported. By feeding the culture of Salinispora tropica NPS21184with 0.1% valeric acid, the concentration of NPI-2080 was increased from0.90 mg/L in the control (no valeric acid addition) to 109.21 mg/L inthe valeric acid-fed culture. While the concentration of SalinosporamideA (II-16) was decreased in the valeric acid-fed culture, it was stillthe major salinosporamide metabolite present in the saline fermentation,with a peak titer at 156.88 mg/L. It is desirable to perform theprecursor-directed or precursor analog-directed biosynthetic studies forthe production of desired analogs in a medium with decreasedconcentration of the major metabolite. In order to investigate theapplication of non-saline, low chloride media in the precursor-directedbiosynthetic study, two seed media and two production media preparedwith the following ingredients, per liter of DI H₂O:

Media components BAS.Sd LoCl.Sd BAS.Pd LoCl.Pd Starch   10 g   10 a   10g   10 g Hy Soy   4 g   4 g   4 g   4 g Yeast Extract   4 g   4 g   4 g  4 g Salt supplements NaCl   24 g 0   24 g 0 Na₂SO₄ 0   40 g 0   20 gKCl 0.69 g 0 0.69 g 0 K₂SO₄ 0   1 g 0   1 g CaCO₃ 0.43 g 0.43 g   1 g  1 g MgSO₄•7H₂O 4.29 g 4.29 g 0 0 CaCl₂•2H₂O 0.43 g 0.43 g 0.43 g 0Fe₂(SO₄)₃   40 mg   40 mg   40 mg   40 mg SrCl₂ 15.5 mg 15.5 mg 15.5 mg15.5 mg H₃BO₃ 21.5 mg 21.5 mg 21.5 mg 21.5 mg NaF  2.6 mg  2.6 mg  2.6mg  2.6 mg

Seed Medium BAS.SD and Production Medium BAS.PD are saline fermentationmedia containing 24 g/L NaCl and other ion supplements. Seed MediumLoCL.SD and Production Medium LoCl.PD are low chloride, sodiumsulfate-based media. Production Medium LoCl.PD contains extremely lowlevels of chloride ion, with the known chloride ion derived from 15.5mg/L SrCl₂ and carryover chloride ion from Hy Soy and yeast extract.

Seed cultures were prepared by inoculating frozen stocks of Salinisporatropica NPS21184 into two different seed media, BAS.SD and LoCl,SD. Thefirst seed cultures (10 mL medium) in 50-mL culture tubes were incubatedfor 3 days before inoculating 5 mL of the first seed culture into thesecond seed medium with the same composition as the first seed medium.The second seed cultures (100 mL medium in 500-mL Erlenmeyer flask) wereincubated for 2 days before inoculating 5 mL of the second seed cultureinto the production media (100 mL in a 500-mL Erlenmeyer flask). BAS.SDseed culture was inoculated into BAS.PD production medium while LoCl.SDseed culture was inoculated into LoCl.PD production medium.

At about 46 hour after inoculation, valeric acid was added to theproduction cultures, except for the control production cultures (novaleric acid) at a final concentration of 0.1%. The production cultureswere incubated for 2 hours before sterile XAD-7 resin slurry was addedto the production cultures in a final concentration of 20 g/L. Theproduction culture was then further incubated and harvested at varioustime points to determine productivity.

3.5 mL of the fermentation culture was mixed with equal volume of EtOAcin an extraction tube to extract the various metabolites from theproduction culture. The mixture was shaken on the shaker for 1 hr. Analiquot of the extract (1 mL) was removed and dried under a stream ofnitrogen. The dried extract was stored at −20° C. freezer before HPLCanalysis. The dried extract was resuspended in 320 μL DMSO and injectedinto HPLC using the 00TF14 method for analysis.

The concentration of Salinosporamide A (II-16), NPI-0047 (II-17),NPI-2065 (II-26) and NPI-2080 (II-30) was monitored by Agilent HP1100HPLC using an ACE C-18 reversed-phase column (4.6×150 mm), and solventsystem consisting of water (0.01% TFA) as solvent A and acetonitrile(0.01% TFA) as solvent B. The elution was started at 100% solvent A for1 min, 100% solvent A to 35% solvent A gradient in 7 min, held at 35%solvent A for 11 min, 35% solvent A to 100% solvent B in 8 min and heldat 100% solvent B for 9 min at a flow rate of 1.5 mL/min with thedetector wavelength set at 210 nm and column temperature 35° C.

The maximum concentrations of Salinosporamide A, NPI-0047, NPI-2065 andNPI-2080 are summarized in the Table below.

TABLE 57 Effect of valeric acid on the production of Salinosporamide A,NP1-0047, NPI-2065 and NPI-2080 in saline and low chloride media Titers(mg/L) Media Salinosporamide NPI-0047 NPI-2065 NPI-2080 conditions A(II-16) (II-17) (II-26) (II-30) BAS.PD 274 6.6 5.5 0.70 BAS.PD + 1310.45 2.9 121 0.1% Valeric acid LoCl.PD 96 25 0.99 1.1 LoCl.PD + 45 8.31.5 145 0.1% Valeric acid

The concentration of Salinosporamide A in the sodium sulfate based withextremely low chloride medium was significantly lower than the salinefermentation condition. The concentration of Salinosporamide A in thelow chloride medium (LoCl.PD) was 96 mg/L, about 35% of the salinefermentation condition. This may due to the lack of the substratechlorine for the production of Salinosporamide A. By feeding valericacid to the low chloride medium LoCl.PD, NPI-2080 was the majorsalinosporamide metabolite in the fermentation at a concentration of 145mg/L, a 130-fold increase in production as compared with the controlLoC.PD medium with no valeric acid. In the saline fermentation mediumwith valeric acid, Salinosporamide A was still the major salinosporamidemetabolite. The low chloride medium (LoCl.PD) is therefore a good mediumfor the precursor directed and precursor-analog directed biosyntheticstudy for production of desired salinosporamide analogs.

Example 24 Seed and Production Media Compositions for Fermentation ofSalinispora tropica Organism

TABLE 58 Seed Medium—Defined Salt Composition Component per liter of DIH₂O Glucose 8 Hy Soy (Kerry Bio- 6 Science, cat. no. 5X59089) YeastExtract (USE 6 Corp. cat. no. 23547) NaCl 24.04 g MgSO₄•7H₂O 4.29 g KBr85.9 mg KCl 686.8 mg CaCO₃ 429.3 mg CaCl₂•2H₂O 429.3 mg SrCl₂ 15.5 mgH₃BO₃ 21.5 mg NaF 2.6 mg NiSO 57.7 μg CoCl₂•6H₂O 207.9 μg

TABLE 59 Seed Medium—“Instant Ocean” Component per liter of DI H₂OGlucose  8 g Hy Soy (Kerry Bio-  6 g Science, cat. no. 5X59089) YeastExtract (USB  6 g Cog). cat. no. 23547) Salt Mixture (Aquarium Systems,“Instant Ocean”) 30 g

TABLE 60 Production Medium—Defined Salt Medium Component per liter of DIH₂O Soluble Starch (USE 10 g Corp. No 21695) Hy Soy (Kerry Bio- 4 gScience, No. 5X59089) Yeast Extract (USE 4 g Corp. No. 23547) CaCO₃ 1 gFe₂(SO₄)₃ 40 mg KBr 100 mg NaCl 24.04 g KCl 686.8 mg CaCl₂•2H₂O 429.3 mgSrCl₂ 15.5 mg H₃BO₃ 21.5 mg NaF 2.6 mg CoCl₂•6H₂O 52 μg NaCl 24 g

TABLE 61 Production Medium—“Instant Ocean” Component per liter of DI H₂OSoluble Starch (USB 10 g Corp. No 21695) Hy Soy (Kerry Bio- 4 g Science,No. 5X59089) Yeast Extract (USB 4 g Corp. No. 23547) CaCO₃ 1 g Fe₂(SO₄)₃40 mg KBr 100 mg Salt Mixture (Aquarium 30 g Systems, “Instant Ocean”)

Example 25 Medium Preparation Procedure

For one liter of medium (seed or production), 500 mL deionized water isadded to a 1 L beaker with a stir bar. The ingredients are added one byone to the beaker of stirring water. After all ingredients are added tothe beaker, an additional 500 mL deionized water is added to the beaker.When all the ingredients are dissolved (no heat is applied in thisprotocol), 100-mL portions of the medium are pipetted into 500 mLErlenmeyer flasks. The flasks are capped with a piece of cheeseclothfollowed by a piece of Bioshield sterile wrap and secured with a rubberband. The flasks are autoclaved at 121° C. for 30 minutes before used.

Example 26 Fermentation of S. tropica Organism

A two stage seed train was used for a portion of this work. For thefirst seed stage, two frozen stock cultures of S. tropica, StrainNPS021184, ATCC accession number PTA-6685, of 1.5 mL each weretransferred to 100 ml of INSTANT OCEAN® seed medium of Example 24 in a500 ml Erlenmeyer flask. This culture was incubated at 28° C. and 250rpm on a rotary shaker. After 3 days, a 5-mL aliquot of the first seedculture was inoculated into a 500-mL Erlenmeyer flask containing 100 mLof the same seed medium. This second seed-stage culture was incubated at28° C. and 250 rpm on a rotary shaker for 2 days. Five-mL of the secondseed-stage culture was then transferred to a 500-mL Erlenmeyer flaskscontaining 100 mL of the INSTANT OCEAN® production medium of Example 24.This production culture was incubated at 28° C. and 250 rpm for 5 days.Samples of the culture were taken at intervals and the dry cell weightwas determined as follows: 5 mL of culture broth was centrifuged at 2300rpm for 20 minutes, decanted retaining the mycelia, and resuspended with5 mL of DI water with vortexing for 10 seconds; the sides of thecentrifuge tube were rinsed with 2 mL of DI water; the suspension wasthen centrifuged an additional 20 minutes at 2300 rpm, the supernatantwas discarded, and the mycelia was dried at 70° C. for at least 72 hoursand weighed.

TABLE 62 GrowthProfile for Example 26. Culture Average dry Average dryAge cell weight (mg) cell weight (mg) (Hours) per 5 ml culture per mlculture 24 17.0 3.4 48 30.0 6.0 72 33.5 6.7 96 34.0 6.8 120 34.0 6.8

Onset of the stationary phase of the fermentation occurred approximatelyat or before 48 hrs after inoculation of the culture.

Example 27 Resin Preparation for Fermentation

Prior to addition to a fermentation culture, the resin, as received, wasprepared by washing, drying, re-slurrying, and autoclaving as follows.Washing: for 1 kg of resin, the resin was washed sequentially with 3liters of DI water, then 1.2 liters of acetone, 1.2 liters of methanol,and 10 liters of DI water. The resin was washed with one liquid and thendrained before adding the next liquid by using a Buchner funnel fittedwith a vacuum flask (apply vacuum to pull the solvent). Drying: afterthe final wash step and the resin is drained by applying vacuum to pullthe visible residual water from the resin. Re-slurry: 200 g of driedresin is added to 400 ml of DI water in a 1 liter bottle. This bottlewith resin is autoclaved at 121° C. for 30 minutes prior to use.

For use, the appropriate amount of sterile resin slurry is transferredto the fermentation vessel at the desired point in the fermentationusing appropriate sterile procedures.

Example 28 Fermentation Sampling and Determination of Production ofDesired Compounds

Sampling: When no resin is present, broth samples are taken for analysisby conventional methods. When resin is present, care must be exercisedto obtain a representative ratio of broth to resin: while swirling thefermentation flask sufficiently to mix the resin in the broth, a 10 mlwide-tip pipette is used to extract a 3.5 ml sample of broth. This brothsample is transferred to a 16×100 mm glass extraction tube (12 mlcapacity). Ethyl acetate (3.5 ml) is added to the broth and mixed for 1hour on a Labquake rotisserie shaker. One ml of the ethyl acetateportion of the extract (top layer), is transferred to a 1.8 ml glassHPLC vial and dried under a stream of nitrogen at room temperature. Thedried extract is stored at −80° C. prior to analysis.

Analysis: The dried extract is resuspended in 320 μL DMSO and 5 μL isinjected into an Agilent HP1100 HPLC fitted with an ACE C-18reversed-phase column (4.6×150 mm). A step gradient of water with 0.01%trifluoroacetic acid and acetonitrile with 0.01% trifluoroacetic acid isused as the mobile phase. The elution profile of Table 1 is used. Columntemperature was 35° C.

TABLE 63 HPLC Elution Profile Solvent A Solvent B Water Acetonitrilewith 0.01% with 0.01% trifluoroacetic trilluoroacetic Time acid acidcomments  0-1 min. 100% 0% isocratic  1-8 min. Start: 100% Start: 0%gradient End: 35% End: 65%  8-19 min. 35% 65% Isocratic 19-27 min.Start: 35% Start: 65% gradient End: 0% End: 100% 27-36 min. 0% 100%isocratic

Detection is with an Agilent photo-diode array variable wavelength UVdetector with the wavelength set at 210 nm.

After equilibrating the column with a mobile phase of water with 0.01%trifluoroacetic acid, the sample is injected. The elution isaccomplished with 100% Solvent A for 1 minute, then with a gradient over7 minutes of 100% Solvent A to 35% Solvent A, then with 35% Solvent Afor 11 minutes, then with a gradient over 8 minutes of 35% Solvent A to0% Solvent A (100% Solvent B), then with 100% Solvent B for 9 minutes.The mobile phase flow rate is 1.5 ml/min; the detector wavelength is 210nm; the column temperature is 35° C.

Example 29 Effect of Time of Addition of the XAD-7 Resin to theFermentation Culture on the Yield of Salinosporamide A

A culture of S. tropica NPS21184 was grown using the chemically definedsalt seed and production media described in Example 24 and following theprocedures described in Examples 25 and 26. To different fermentationflasks, XAD-7 resin was added at 24, 48, 72, and 96 hours afterinoculation following the procedure of Example 27. The resinconcentration was 20 g/l. A control fermentation had no resin added. Thefermentations were sampled every 24 hours for production ofSalinosporamide A. The fermentations were terminated at 144 h after thefinal samples were taken. The results are shown below. Analysis forSalinosporamide A was performed as described in Example 28.

TABLE 64 Salinosporamide Concentration at Different Times in theFermentation Time of resin Salinosporamide A (mg/L) addition 24 h 48 h72 h 96 h 120 h 144 h No 0 3 4 2 0.1 0.1 addition 24 h 0 62 233 275 245222 48 h ND 3 158 224 219 178 72 h ND ND 4 108 99 92 96 h ND ND ND 2 1510 ND = Not determined

Maximum product concentrations were achieved at 96 hours. Addition ofresin at hour 24 resulted in the highest concentration of product. Basedon the results of Example 26, addition of the resin at 24 hr would beduring the exponential phase, prior to the onset of the stationary phaseof growth. Addition at 48 hr would approximately coincide with the onsetof the stationary phase. Later additions were likely after the onset ofthe stationary phase.

TABLE 65 Increase in Maximum Product Titer for Different Resin AdditionTimes Compared to Control Difference in titer Amount of Time of Maximumas compared NPI-0052 synthesized resin titer to 24 h resin since resinaddition (mg/L) addition (mg/L) addition at 24 h (mg/L) 24 h 275 — — 48h 224 51 62 72 h 108 167 158

The production with resin added at hour 24 resulted in a 69-foldincrease in production over the control condition.

Example 30 Effect of the Quantity and Type of Resin Used on the Yield ofSalinosporamide A: XAD-2, XAD-4, XAD-7, and XAD-16

A culture of S. tropica NPS21184 was grown using the INSTANT OCEAN® seedand production media described in Example 24 and following theprocedures described in Examples 25 and 26. At 24 hours afterinoculation, XAD-2, XAD-4, XAD-7 or XAD-16 resin was added to separateflasks at 20, 30 or 40 g/l concentration following the method of Example27. A control flask had no resin added. The cultures were maintained foranother 72 hours. Final samples were taken and analyzed forSalinosporamide A according to Example 28. The results are presentedbelow:

TABLE 66 Salinosporamide A Concentrations for Different Resins andDifferent Resin Concentrations Increase in NPI- Final NPI-0052 0052production Concentration titer as compared to Resin of resin (g/L)(mg/L) the control Control (no resin) 0 5.7 — XAD-2 20 266 47 fold 30297 52 fold 40 290 51 fold XAD-4 20 207 36 fold 30 213 37 fold 40 232 41fold XAD-7 20 278 49 fold 30 345 61 fold 40 316 55 fold XAD-16 20 218 38fold 30 237 42 fold 40 171 30 fold

The use of acrylic XAD-7 resulted in the highest production of theSalinosporamide A, with up to a 61-fold improvement over the controlcondition. The SDVB resins also increased the production of NPI-0052with XAD-2 being the most effective SDVB resin showing up to a 52-foldincrease over the control. While not being bound by any particulartheory, the higher polarity of the acrylic XAD-7 resin may explain whyit was the best resin tested in this study for the increase inproduction of NPI-0052. Resins with large surface area are lesseffective in stabilization of NPI-0052 as XAD-4, and XAD-16 are lesseffective than XAD-2 in increasing the production of NPI-0052.

Example 31 Comparison of XAD-7, IRA-67, IRC-50 and IRP-64 on Productionof Salinosporamide A

A culture of S. tropica NPS21184 was grown in the INSTANT OCEAN® seedand production medium of Example 24, following the procedures ofExamples 25 and 26. At 24 hours after inoculation, XAD-7, IRA-67, IRC-50or IRP-64 resin was added to separate flasks at 20, 30 or 40 g/lconcentration following the procedure of Example 27. A control flask hadno resin added. The cultures were maintained for another 72 hours. Finalsamples were taken and analyzed for Salinosporamide A according toExample 28. The results are presented below:

TABLE 66 Salinosporamide A Concentrations for Different Resins andDifferent Resin Concentrations Increase in NPI- Final NPI-0052 0052production Concentration titer as compared to Resin of resin (g/L)(mg/L) the control Control (no resin) 0 3.9 — XAD-7 10 190 49 fold 20272 70 fold 30 309 79 fold 40 321 82 fold IRA-67 10 0.2 (Decrease) 20 0(Decrease) 30 0 (Decrease) 40 0 (Decrease) IRC-50 10 0 (Decrease) 20 0(Decrease) 30 0 (Decrease) 40 0 (Decrease) IRP-64 10 0 (Decrease) 20 0(Decrease) 30 0 (Decrease) 40 0 (Decrease)

Addition of Amberlite IRA-67, Amberlite IRC-50 and Amberlite IRP-64 tothe cultures of S. tropica inhibited the production of SalinosporamideA. Amberlite XAD-7 enhanced the production of this compound by 82-foldwith the highest product concentration when 40 g/l of resin was used.

Example 32 Comparison of HP-20, HP-2MG. SP-207 and SP-850 for theProduction of Salinosporamide A

A culture of S. tropica NPS21184 was grown in the INSTANT OCEAN® seedand production medium of Example 24, following the procedures ofExamples 25 and 26. At 24 hours after inoculation, HP-20, HP-2MG, SP-207or SP-850 resin was added to separate flasks at 20, 30 or 40 g/lconcentration following the procedure of Example 27. A control flask hadno resin added. The cultures were maintained for another 72 hours. Finalsamples were taken and analyzed for Salinosporamide A according toExample 28. The results are presented below:

TABLE 67 Salinosporamide A Concentrations for Different Resins andDifferent Resin Concentrations Increase in NPI- Final NPI-0052 0052production Concentration titer as compared to Resin of resin (g/L)(mg/L) the control Control (no resin) 0 3.7 — Diaion HP-20 10 219 59folds 20 293 79 folds 30 309 84 folds 40 286 77 folds Diaion HP-2MG 10162 44 folds 20 217 59 folds 30 250 68 folds 40 250 68 folds SepabeadsSP-207 10 201 54 folds 20 165 45 folds 30 146 40 folds 40 201 54 foldsSepabeads SP-805 10 208 56 folds 20 246 67 folds 30 261 71 folds 40 22561 folds

All of these resins enhanced the production of Salinosporamide A. Thehighest production came from the HP-20 resin with a production increasesimilar to XAD-7.

Example 33 Production of Compounds 1, 5, 6, 7, and 9 from Strain CNB476with XAD-7 Resin Present

Microorganism S. tropica, strain CNB476 (ATCC accession numberPTA-5275), was grown in a 500-ml flask containing 100 ml of vegetativemedium consisting of the following per liter of deionized water:glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; and syntheticsea salt (Instant Ocean, Aquarium Systems), 30 g. The first seed culturewas incubated at 28 degree C for 3 days on a rotary shaker operating at250 rpm. Five ml each of the first seed culture was inoculated intothree 500-ml flasks containing of 100 ml of the vegetative medium. Thesecond seed cultures were incubated at 28 degree C and 250 rpm on arotary shaker for 2 days. Five ml each of the second seed culture wasinoculated into thirty-five 500-ml flasks containing of 100 ml of thevegetative medium. The third seed cultures were incubated at 28 degreeand 250 rpm on a rotary shaker for 2 days. Five ml each of the thirdseed culture was inoculated into four hundred 500-ml flasks containing100 ml of the Production Medium C consisting of the following per literof deionized water: starch, 10 g; yeast extract, 4 g; Hy-Soy, 4 g;ferric sulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1g; and synthetic sea salt (INSTANT OCEAN®), 30 g. The productioncultures were incubated at 28 degree C and 250 rpm on rotary shakers for1 day. Approximately 2 to 3 grams of sterile Amberlite XAD-7 resin wereadded to the production cultures. The production cultures were furtherincubated at 28 degree C and 250 rpm on rotary shakers for 5 days andachieved a titer of Compound 1 of about 200 mg/L. The culture broth wasfiltered through cheese cloth to recover the Amberlite XAD-7 resin. Theresin was extracted 2 times with 6 liters ethyl acetate followed by 1time with 1.5 liters ethyl acetate. The combined extracts were dried invacuo. The dried extract, containing 3.8 grams of Compound 1 and lesserquantities of Compounds 5 and 6, was then processed for the recovery ofCompounds 1, 3, 5, 6, 7, and 9.

Example 34 Production of Compounds 1, 3, 5, 6, 7, and 9 from StrainNPS21184 with XAD-7 Resin Present

Microorganism S. tropica, strain NPS21184, was grown in a 500-ml flaskcontaining 100 ml of vegetative medium consisting of the following perliter of deionized water: glucose, 8 g; yeast extract, 6 g; Hy-Soy, 6 g;and synthetic sea salt (INSTANT OCEAN®), 30 g. The first seed culturewas incubated at 28 degree C for 3 days on a rotary shaker operating at250 rpm. Five ml of the first seed culture was inoculated into 500-mlflask containing of 100 ml of the vegetative medium. The second seedcultures were incubated at 28 degree C and 250 rpm on a rotary shakerfor 2 days. Five ml each of the second seed culture was inoculated into500-ml flask containing of 100 ml of the vegetative medium. The thirdseed cultures were incubated at 28 degree and 250 rpm on a rotary shakerfor 2 days. Five ml each of the third seed culture was inoculated into500-ml flask containing 100 ml of Production Medium D consisting of thefollowing per liter of deionized water: starch, 20 g; yeast extract, 4g; Hy-Soy, 8 g; ferric sulfate, 40 mg; potassium bromide, 100 mg;calcium carbonate, 1 g; and synthetic sea salt (INSTANT OCEAN®), 30 g.The production cultures were incubated at 28 degree C and 250 rpm onrotary shakers for 1 day. Approximately 2 to 3 grams of sterileAmberlite XAD-7 resin were added to the production culture. Theproduction culture was further incubated at 28 degree C and 250 rpm onrotary shaker for 4 days and achieved a titer of 350-400 mg/L forCompound 1.

Alternatively, the production of the compounds can be achieved in a 42 Lfermentor system using strain NPS21184. Strain NPS21184 was grown in a500-ml flask containing 100 ml of vegetative medium consisting of thefollowing per liter of deionized water: glucose, 8 g; yeast extract, 6g; Hy-Soy, 6 g; and synthetic sea salt (INSTANT OCEAN®), 30 g. The firstseed culture was incubated at 28 degree C for 3 days on a rotary shakeroperating at 250 rpm. Five ml of the first seed culture was inoculatedinto 500-ml flask containing of 100 ml of the vegetative medium. Thesecond seed cultures were incubated at 28 degree C and 250 rpm on arotary shaker for 2 days. Twenty ml each of the second seed culture wasinoculated into 2.8 L Fernbach flask containing 400 ml of the vegetativemedium. The third seed cultures were incubated at 28 degree and 250 rpmon a rotary shaker for 2 days. 1.2 L of the third seed culture wasinoculated into a 42 L fermentor containing 26 L of Production Medium C.Production Medium D and Production Medium E, with the followingcomposition, can also be used. Production Medium E consisting of thefollowing per liter of deionized water: starch, 15 g; yeast extract 6 g;Hy-Soy, 6 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calciumcarbonate, 1 g; and synthetic sea salt (Instant Ocean, AquariumSystems), 30 g. The fermentor cultures were operated at the followingparameters: temperature, 28 degree C; agitation, 200 rpm; aeration, 13L/min and back pressure, 4.5 psi. At 36 to 44 hours of the productioncycle, approximately 600 grams of sterile Amberlite XAD-7 resin wereadded to the fermentor culture. The production culture was furtherincubated at the above operating parameters until day 4 of theproduction cycle. The aeration rate was lowered to 8 L/min. At day 5 ofthe production cycle, the fermentor culture achieved a titer of about300 mg/L for Salinosporamide A. The culture broth was filtered throughcheese cloth to recover the Amberlite XAD-7 resin. The resin wasextracted with 2 times 4.5 L liters ethyl acetate followed by 1 time 1.5liters ethyl acetate. The combined extracts were dried in vacuo. Thedried extract was then processed for the recovery of Compounds 1, 2, 3,5, 6, and 9.

Example 35 Purification of Compounds Isolated in Examples 33 and 34

The pure Compounds of 1, 5, 6, 7, and 9 were obtained by flashchromatography followed by HPLC. Eight grams crude extract containing3.8 grams of Compound 1 and lesser quantities of Compounds 3, 6, 7, and9 was processed by flash chromatography using Biotage Flash40i systemand Flash 40M cartridge (KP-Sil Silica, 32-63 μm, 90 grams). The flashchromatography was developed by the following step gradient:

1. Hexane (1 L)

2. 10% Ethyl acetate in hexane (1 L)

3. 20% Ethyl acetate in hexane, first elution (1 L)

4. 20% Ethyl acetate in hexane, second elution (1 L)

5. 20% Ethyl acetate in hexane, third elution (1 L)

6. 25% Ethyl acetate in hexane (1 L)

7. 50% Ethyl acetate in hexane (1 L)

8. Ethyl acetate (1 L)

Fractions containing Compound 1 in greater or equal to 70% UV purity byHPLC were pooled and subject to HPLC purification, as described below,to obtain Compound 1, along with Compounds 5 and 6, each as purecompounds.

TABLE 68 Column Phenomenex Luna 10 u Silica Dimensions 25 cm X 21.2 mmID Flow rate 25 ml/min Detection ELSD Solvent Gradient of 24%EtOAc/hexane for 19 min, 24% EtOAc/hexane to 100% EtOAc in 1 min, then100% EtOAc for 4 min

The fraction enriched in Compound 1 (described above; ˜70% pure withrespect to Compound 1) was dissolved in acetone (60 mg/ml). Aliquots(950 μl) of this solution were injected onto a normal-phase HPLC columnusing the conditions described above. Compound 1 typically eluted after14 minutes and Compounds 6 and 7 co-eluted as a single peak at 11 min.When parent samples containing Compounds 2, 5, and 9 were processed,Compound 2 eluted at 22 minutes, while Compounds 5 and 9 co-eluted at 23minutes during the 100% ethyl acetate wash. Fractions containingCompound 1 and minor analogs were pooled based on composition ofcompounds present, and evaporated under reduced pressure on a rotaryevaporator. This process yielded pure Compound A, as well as separatefractions containing minor Compounds 5, 6, 7, and 9, which were furtherpurified as described below.

Sample containing Compounds 6 and 7 generated from the process describedabove were further separated using reversed-phase preparative HPLC asfollows. The sample containing compound 6 (70 mg) was dissolved inacetonitrile at a concentration of 10 mg/ml, and 500 μl was loaded on anHPLC column of dimensions 21 mm i.d. by 15 cm length containing EclipseXDB-C18 support. The solvent gradient increased linearly from 15%acetonitrile/85% water to 100% acetonitrile over 23 minutes at a flowrate of 14.5 ml/min. The solvent composition was held at 100%acetonitrile for 3 minutes before returning to the starting solventmixture. Compound 7 eluted at 17.5 minutes while Compound 6 eluted at 19minutes under these conditions.

Crystalline Compound 7 was obtained using a vapor diffusion method.Compound 7 (15 mg) was dissolved in 100 μl of acetone in a 1.5 mlv-bottom HPLC vial. This vial was then placed inside a larger sealedvessel containing 1 ml of pentane. Crystals suitable for X-raycrystallography experiments were observed along the sides and bottom ofthe inner vial after 48 hours of incubation at 4° C. Crystallographydata was collected on a Bruker SMART APEX CCD X-ray diffractometer(F(000)=2656, Mo_(Kα) radiation, λ=0.71073 Å, μ=0.264 mm⁻¹, T=100K) atthe UCSD Crystallography Lab and the refinement method used wasfull-matrix least-squares on F². Crystal data NPI-2065: C₁₅H₂₀ClNO₄,MW=313.77, tetragonal, space group P4(1)2(1)2, a= b=11.4901(3) Å,c=46.444(2) Å, α=β=γ=90°, vol=6131.6(3) Å³, Z=16, ρ_(calcd)=1.360 g cm³,crystal size, 0.30×0.15×0.07 mm³, θ range, 1.75-26.00°, 35367reflections collected, 6025 independent reflections (R_(int)=0.0480),final R indices (I>2σ(I)): R₁=0.0369, wR₂=0.0794, GOF=1.060.

In order to separate Compounds 9 from 5, a reverse-phase isocraticmethod was employed. Sample (69.2 mg) containing both compounds wasdissolved in acetonitrile to a concentration of 10 mg/ml, and 500 μl wasloaded on a reverse-phase HPLC column (ACE 5 C18-HL, 15 cm×21 mm ID) perinjection. An isocratic solvent system of 27% acetonitrile/63% water atflow rate of 14.5 ml/min was used to separate compounds II-28 and II-20,which eluted after 14 and 16 minutes, respectively. Fractions containingcompounds of interest were immediately evaporated under reduced pressureat room temperature on a rotary evaporator. Samples were then loadedonto a small column of silica and eluted with 10 ml of 70% hexane/30%acetone to remove additional impurities.

Samples generated from the preparative normal-phase HPLC methoddescribed above that contained Compound 5, but which were free ofCompound 9 could also be triturated with 100% EtOAc to remove minorlipophilic impurities.

Compound 1: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. Low Res. Mass: m/z314 (M+H), 336 (M+Na).

Compound 5: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. Low Res. Mass: m/z266 (M+H); HRMS (ESI), m/z 266.1396 (M+H), Δ_(calc)=1.2 ppm.

Compound 6: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. Low Res. Mass: m/z328 (M+H), 350 (M+Na); HRMS (ESI), m/z 328.1309 (M+H), Δ_(calc)=−2.0ppm, C₆H23NO₄Cl.

Compound 7: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm; HRMS (ESI), m/z314.1158 (M+H), Δ_(calc)=−0.4 ppm, C₁₅H₂₁NO₄Cl.

Compound 9: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm; HRMS (ESI), m/z266.1388 (M+H), Δ_(calc)=−1.8 ppm, C₁₄H₂₀NO₄.

3B: Purification of Compound of Formula I-7

A Biotage Flash 75Li system with a Flash 75 L KP-Sil cartridge was usedto process the filtered crude extract (10.0 g), enriched in Compound 1and containing Compound of Formula I-7. The crude extract was dissolvedto a concentration of 107 mg/ml in acetone and loaded directly onto thecartridge. The following solvent step gradient was then run through thecartridge at a flow rate between 235 ml/min and 250 ml/min

1. 10% EtOAc in n-Heptane (3.2 L)

2. 25% EtOAc in n-Heptane (16 L)

3. 30% EtOAc in n-Heptane (5.4 L)

Fractions enriched in Compound 1 were pooled and concentrated byrotavapor until ˜5% of the total pooled volume of solvent remained. Thesolvent was removed, leaving behind the white solid.

A crystallization was then performed on the solid by dissolving thesample (4.56 g) in 1:1 acetone:n-heptane (910 ml). The solvent wasslowly evaporated using a rotary evaporator until the solvent wasreduced to about 43% of its original volume. The solution (supernatant)was removed and concentrated (598 mg).

The supernatant was dissolved in acetone (80 mg/ml). Aliquots (500 μl)of this solution were injected onto a normal-phase HPLC column using theconditions described above for normal phase purification of Compounds 1,6, 7, and 9. Compound 3 eluted at 7.5 minutes as a pure compound.

Compound 3: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. Low Res. Mass: m/z298 (M+H), 320 (M+Na).

Example 36 Fermentation of Compounds 2, 4, and 8

Microorganism S. tropica, strain CNB476, was grown in a 500-ml flaskcontaining 100 ml of the first vegetative medium consisting of thefollowing per liter of deionized water: glucose, 4 g; Bacto tryptone, 3g; Bacto casitone, 5 g; and synthetic sea salt (Instant Ocean, AquariumSystems), 30 g. The first seed culture was incubated at 28 degree C for3 days on a rotary shaker operating at 250 rpm. Five ml of the firstseed culture was inoculated into a 500-ml flask containing 100 ml of thesecond vegetative medium consisting of the following per liter ofdeionized water: starch, 10 g; yeast extract, 4 g; peptone, 2 g; ferricsulfate, 40 mg; potassium bromide, 100 mg; calcium carbonate, 1 g; andsodium bromide, 30 g. The second seed cultures were incubated at 28° C.for 7 days on a rotary shaker operating at 250 rpm. Approximately 2 to 3gram of sterile Amberlite XAD-7 resin were added to the second seedculture. The second seed culture was further incubated at 28° C. for 2days on a rotary shaker operating at 250 rpm. Five ml of the second seedculture was inoculated into a 500-ml flask containing 100 ml of thesecond vegetative medium. The third seed culture was incubated at 28° C.for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to 3gram of sterile Amberlite XAD-7 resin were added to the third seedculture. The third seed culture was further incubated at 28° C. for 2days on a rotary shaker operating at 250 rpm. Five ml of the thirdculture was inoculated into a 500-ml flask containing 100 ml of thesecond vegetative medium. The fourth seed culture was incubated at 28°C. for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to3 gram of sterile Amberlite XAD-7 resin were added to the fourth seedculture. The fourth seed culture was further incubated at 28° C. for 1day on a rotary shaker operating at 250 rpm. Five ml each of the fourthseed culture was inoculated into ten 500-ml flasks containing 100 ml ofthe second vegetative medium. The fifth seed cultures were incubated at28° C. for 1 day on a rotary shaker operating at 250 rpm. Approximately2 to 3 grams of sterile Amberlite XAD-7 resin were added to the fifthseed cultures. The fifth seed cultures were further incubated at 28° C.for 3 days on a rotary shaker operating at 250 rpm. Four ml each of thefifth seed culture was inoculated into one hundred and fifty 500-mlflasks containing 100 ml of the production medium having the samecomposition as the second vegetative medium. Approximately 2 to 3 gramsof sterile Amberlite XAD-7 resin were also added to the productionculture. The production cultures were incubated at 28° C. for 6 day on arotary shaker operating at 250 rpm. The culture broth was filteredthrough cheese cloth to recover the Amberlite XAD-7 resin. The resin wasextracted with 2 times 3 liters ethyl acetate followed by 1 time 1 literethyl acetate. The combined extracts were dried in vacuo. The driedextract, containing 0.42 g of the compound Formula II-17 and 0.16 gramthe compound of Formula II-18, was then processed for the recovery ofthe compounds.

Example 37 Purification of Compounds 2, 4, and 8

Pure Compounds 2 and 4 were obtained by reversed-phase HPLC as describedbelow:

TABLE 69 Column ACE 5 C18-HL Dimensions 15 cm X 21 mm ID Flow rate 14.5ml/min Detection 214 nm Solvent Gradient of 35% Acetonitrile/65% H₂O to90% Acetonitrile/10% H₂O over 15 min

Crude extract (100 mg) was dissolved in 15 ml of acetonitrile. Aliquots(900 μl) of this solution were injected onto a reversed-phase HPLCcolumn using the conditions described above. Compounds 2 and 4 eluted at7.5 and 9 minutes, respectively. Fractions containing the pure compoundswere first concentrated using nitrogen to remove organic solvent. Theremaining solution was then frozen and lyophilized to dryness.

An alternative purification method for Compounds 2 and 4 was developedfor larger scale purification and involved fractionation of the crudeextract on a normal phase VLC column. Under these conditions, sufficientamounts of several minor metabolites were identified, including Compound8. The crude extract (2.4 g) was dissolved in acetone (10 ml) and thissolution adsorbed onto silica gel (10 cc) by drying in vacuo. Theadsorbed crude extract was loaded on a normal phase silica VLC column(250 cc silica gel, column dimensions 2.5 cm diameter by 15 cm length)and washed with a step gradient of hexane/EtOAc, increasing in thepercentage of hexane in steps of 5% (100 ml solvent per step). Themajority of compound 1 eluted in the 60% hexane/40% EtOAc wash while themajority of compound 2 eluted in the 50% hexane/50% ethyl acetate wash.Final separation of the compounds was achieved using C18 HPLCchromatography (ACE 5μ C18-HL, 150 mm×21 mm ID) using an isocraticsolvent system consisting of 35% ACN/65% H2O. Under these conditions,Compound 8 eluted at 11 minutes, Compound 2 eluted at 12.00 minutes,traces of Compound 1 eluted at 23.5 minutes, and Compound 4 eluted at25.5 minutes. The resulting samples were dried in vacuo using no heat toremove the aqueous solvent mixture. The spectroscopic data for thesesamples of Compound 1 and Compound 4 were found to be identical withthose of samples prepared from earlier purification methods. The sampleof Compound 4 was found to contain 8% of the lactone hydrolysis productand was further purified by washing through a normal phase silica plug(1 cm diameter by 2 cm height) and eluting using a solvent mixture of20% EtOAc/80% Hexanes (25 ml). The resulting sample was found to containpure Compound 4.

The fractions containing Compound 8 described above were furtherpurified using normal phase semipreparative HPLC (Phenomenex Luna Si10μ, 100 Å; 250×10 mm id) using a solvent gradient increasing from 100%hexane to 100% EtOAc over 20 minutes with a flowrate of 4 ml/min.Compound 8 eluted as a pure compound after 11.5 minutes (0.8 mg, 0.03%isolated yield from dried extract weight).

Compound 2: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. High Res. Mass(APCI): m/z 280.156 (M+H), Δ_(calc)=2.2 ppm, C₁₅H₂₂NO₄.

Compound 4: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. High Res. Mass(APCI): m/z 358.065 (M+H), Δ_(calc)=−1.9 ppm, C₁₅H₂₁NO₄Br.

Compound 8: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm; MS (HR-ESI), m/z280.1556 (M+H) Δ_(calc)=2.7 ppm (C₁₅H₂₂NO₄).

1. A growth medium for use in fermenting a marine microorganism, themedium comprising: (a) Potassium ion at a concentration between 5mMole/L and 13 mMole/L; (b) Calcium species at a concentration between1.8 mMole/L and 6.3 mMole/L; (c) Strontium ion at a concentrationbetween 0.03 mMole/L and 0.32 mMole/L; (d) Borate species at aconcentration between 0.16 mMole/L and 0.81 mMole/L; and (e) Fluorideion at a concentration between 0.02 mMole/L and 0.11 mMole/L.
 2. Thegrowth medium of claim 1, wherein the medium comprises NaCl at aconcentration between 5 g/L and 30 g/L.
 3. The growth medium of claim 1,wherein the medium comprises sulfate species at a concentration between0.07M and 0.28M.
 4. The growth medium of claim 1, wherein the mediumcomprises cobalt ion at a concentration between 0.2 μMole/L and 0.5μMole/L.
 5. The growth medium of claim 1, wherein the medium comprisescobalt ion at a concentration between 1.1 μMole/L and 1.9 μMole/L. 6.The growth medium of claim 1, wherein the medium comprises: (a)Magnesium species at a concentration between 8 mMole/L and 24 mMole/Lg/L; (b) Bromide ion at a concentration between 0.6 mMole/L and 0.8mMole/L; and (c) Carbonate species at a concentration between 2 mMole/Land 10 mMole/L.
 7. The growth medium of claim 1, wherein the mediumcomprises vitamin B₁₂ at a concentration between 0.05 mg/L and 5 mg/L.8. A growth medium for use in fermenting a marine microorganism themedium comprising cobalt ion at a concentration between 0.2 μMole/L and1.9 μMole/L.
 9. The growth medium of claim 8, wherein the cobalt ionconcentration is between 0.2 μMole/L and 0.5 μMole/L.
 10. The growthmedium of claim 8, wherein the cobalt ion concentration is between 0.4μMole/L and 2 μMole/L.
 11. The growth medium of claim 8, wherein themedium comprises a carbon source.
 12. The growth medium of claim 8,wherein the medium comprises a nitrogen source.
 13. The growth medium ofclaim 8, wherein the medium comprises: (a) Potassium ion at aconcentration between 5 mMole/L and 13 mMole/L; (b) Calcium species at aconcentration between 1.8 mMole/L and 6.3 mMole/L; (c) Strontium ion ata concentration between 0.03 mMole/L and 0.3 mMole/L; (d) A boratespecies at a concentration between 0.16 mMole/L and 0.8 mMole/L; and (e)Fluoride at a concentration between 0.02 mMole/L and 0.1 mMole/L, (f) Amagnesium species at a concentration between 8 mMole/L and 24 mMole/L;(g) Bromide ion at a concentration between 0.6 mMole/L and 0.8 mMole/L;and (h) Carbonate species at a concentration at a concentration between2 mMole/L and 10 mMole/L.
 14. The growth medium of claim 8, wherein themedium comprises NaCl at a concentration between 5 g/L and 30 g/L. 15.The growth medium at claim 8, wherein the medium comprises a sulfatespecies at a concentration between 0.07M and 0.3M.
 16. The growth mediumat claim 8, wherein the medium comprises a mixture of NaCl and Na₂SO₄.17. The growth medium at claim 8, wherein the medium comprises vitaminB₁₂ at a concentration between 0.05 mg/L and 5 mg/L. 18-54. (canceled)55. The growth medium at claim 1, wherein the calcium species isselected from the group consisting of calcium ions, calcium bicarbonate,calcium carbonate, calcium bicarbonate calcium sulfate, calciumbisulfate, and a calcium salt; and the borate species is selected fromthe group consisting of borate ion, borate acid, protonated borate ions,potassium borate, sodium borate, dihydrogen borate, hydrogen borate, andborate salt.
 56. The growth medium of claim 3, wherein the sulfatespecies is selected from the group consisting of sulfate ions, bisulfateions, sulfate salts, bisulfate salts, potassium sulfate, sodium sulfate,calcium sulfate, potassium sulfate, potassium bisulfate, sodiumbisulfate, and magnesium sulfate.
 57. The growth medium of claim 6,wherein the magnesium species is selected from the group consisting ofmagnesium ion, magnesium sulfate, magnesium carbonate, magnesiumbicarbonate, magnesium chloride, and magnesium salt, the carbonatespecies is selected from the group consisting of carbonate ion,bicarbonate ion, calcium carbonate, calcium bicarbonate, magnesiumcarbonate magnesium bicarbonate, sodium carbonate, sodium bicarbonate,potassium carbonate, potassium bicarbonate, carbonate salt, andbicarbonate salt.
 58. The growth medium at claim 8, wherein the calciumspecies is selected from the group consisting of calcium ions, calciumbicarbonate, calcium carbonate, calcium bicarbonate, calcium sulfate,calcium bisulfate, and a calcium salt; the borate species is selectedfrom the group consisting of borate ion, boric acid, protonated borateions, potassium borate, sodium borate, dihydrogen borate, hydrogenborate, and borate salt; the magnesium species is selected from thegroup consisting of magnesium ion, magnesium sulfate, magnesiumcarbonate, magnesium bicarbonate, magnesium chloride, and magnesiumsalt; and the carbonate species is selected from the group consisting ofcarbonate ion, bicarbonate ion, calcium carbonate, calcium bicarbonate,magnesium carbonate, magnesium bicarbonate, sodium carbonate, sodiumbicarbonate, potassium carbonate, potassium bicarbonate, carbonate salt,and bicarbonate salt.
 59. The growth medium of claim 15, wherein thesulfate species is selected from the group consisting of sulfate ions,bisulfate ions, sulfate salts, bisulfate salts, potassium sulfate,sodium sulfate, calcium sulfate, potassium sulfate, potassium bisulfate,sodium bisulfate, and magnesium sulfate.